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Aktuelle wissenschaftliche Fachartikel der
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Resolution-adapted recombination of structural features significantly improves sampling in restraint-guided structure calculation
Abstract Recent work has shown that NMR structures can be determined by integrating sparse NMR data with structure prediction methods such as Rosetta. The experimental data serve to guide the search for the lowest energy state towards the deep minimum at the native state which is frequently missed in Rosetta de novo structure calculations. However, as the protein size increases, sampling again becomes limiting; for example, the standard Rosetta protocol involving Monte Carlo fragment insertion starting from an extended chain fails to converge for proteins over 150 amino acids even with guidance from chemical shifts (CS-Rosetta) and other NMR data. The primary limitation of this protocolāthat every folding trajectory is completely independent of every otherāwas recently overcome with the development of a new approach involving resolution-adapted structural recombination (RASREC). Here we describe the RASREC approach in detail and compare it to standard CS-Rosetta. We show that the improved sampling of RASREC is essential in obtaining accurate structures over a benchmark set of 11 proteins in the 15-25 kDa size range using chemical shifts, backbone RDCs and HN -HN NOE data; in a number of cases the improved sampling methodology makes a larger contribution than incorporation of additional experimental data. Experimental data are invaluable for guiding sampling to the vicinity of the global energy minimum, but for larger proteins, the standard Rosetta fold-from-extended-chain protocol does not converge on the native minimum even with experimental data and the more powerful RASREC approach is necessary to converge to accurate solutions. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Effects of point mutations in pVHL on the binding of HIF-1Ī±
Abstract The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans -4-prolyl hydroxylation and is subsequently recognised by the Ī²-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1Ī± and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1Ī± interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization
Abstract Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Crystal structure of bacteriophage ĻNIT1 zinc peptidase PghP that hydrolyzes Ī³-glutamyl linkage of bacterial poly-Ī³-glutamate
Abstract Poly-Ī³-glutamate hydrolase P (PghP) of Bacillus subtilis bacteriophage Ī¦NIT1 hydrolyzes the Ī³-glutamyl peptide linkage of extracellular poly-Ī³-glutamate produced by bacilli, which facilitates infection and propagation of phage progenies. Crystal structure of PghP was determined at a resolution of 1.9 Ć
. Structure of PghP was elucidated as a globular protein with an open Ī±/Ī² mixed core structure and a seven-stranded parallel/anti-parallel Ī²-sheet. The Ī²-sheet contained a core four-stranded parallel Ī²-sheet. A zinc-binding motif, His-Glu-His, was identified at the C-terminal end of the Ī²-sheet. Structure analysis demonstrated that PghP, which had not been previously classified into any peptidase/protease family due to lack of amino acid sequence similarity with known enzymes, had a catalytic center containing a zinc ion and an overall topology resembling mammalian carboxypeptidase A and related enzymes. Structural comparisons indicated important amino acid residues of PghP for catalysis and recognition of the Ī³-peptide bond of poly-Ī³-glutamate, which was confirmed by site-directed mutagenesis of PghP. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: The stabilizing effect of membrane-mimetics on the acquired fold
Abstract A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an amino-terminal signal peptide. In Escherichia coli , typically signal peptides that guide preproteins into the SecYEG protein conduction channel are subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution nuclear magnetic resonance. We employed a soluble form of signal peptidase, which laks the two transmembrane domains (SPase I Ī2-75), and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of a weak peptide-enzyme complex formation. The peptide adopts a U-turn shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable with that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and based on PRE (paramagnetic relaxation enhancement) experiments are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Geofold: Topology-based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability
Abstract Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
A divide-and-conquer approach to determine the Pareto frontier for optimization of protein engineering experiments
Abstract In developing improved protein variants by site-directed mutagenesis or recombination, there are often competing objectives that must be considered in designing an experiment (selecting mutations or breakpoints): stability versus novelty, affinity versus specificity, activity versus immunogenicity, and so forth. Pareto optimal experimental designs make the best trade-offs between competing objectives. Such designs are not ādominatedā; that is, no other design is better than a Pareto optimal design for one objective without being worse for another objective. Our goal is to produce all the Pareto optimal designs (the Pareto frontier), to characterize the trade-offs and suggest designs most worth considering, but to avoid explicitly considering the large number of dominated designs. To do so, we develop a divide-and-conquer algorithm, Protein Engineering Pareto FRontier (PEPFR ), that hierarchically subdivides the objective space, using appropriate dynamic programming or integer programming methods to optimize designs in different regions. This divide-and-conquer approach is efficient in that the number of divisions (and thus calls to the optimizer) is directly proportional to the number of Pareto optimal designs. We demonstrate PEPFR with three protein engineering case studies: site-directed recombination for stability and diversity via dynamic programming, site-directed mutagenesis of interacting proteins for affinity and specificity via integer programming, and site-directed mutagenesis of a therapeutic protein for activity and immunogenicity via integer programming. We show that PEPFR is able to effectively produce all the Pareto optimal designs, discovering many more designs than previous methods. The characterization of the Pareto frontier provides additional insights into the local stability of design choices as well as global trends leading to trade-offs between competing criteria. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Camps 2.0: Exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins
Abstract Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequenceāstructure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to Ī±-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ā¼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/ . Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Characterization of an alternative low energy fold for bovine Ī±-lactalbumin formed by disulfide bond shuffling
Abstract Bovine Ī±-lactalbumin (Ī±LA) forms a misfolded disulfide bond shuffled isomer, X-Ī±LA. This X-Ī±LA isomer contains two native disulfide bridges (Cys 6āCys 120 and Cys 28āCys 111) and two non-native disulfide bridges (Cys 61āCys 73 and Cys 77āCys 91). MD simulations have been used to characterize the X-Ī±LA isomer and its formation via disulfide bond shuffling and to compare it with the native fold of Ī±LA. In the simulations of the X-Ī±LA isomer the structure of the Ī±-domain of native Ī±LA is largely retained in agreement with experimental data. However, there are significant rearrangements in the Ī²-domain, including the loss of the native Ī²-sheet and calcium binding site. Interestingly, the energies of X-Ī±LA and native Ī±LA in simulations in the absence of calcium are closely similar. Thus, the X-Ī±LA isomer represents a different low energy fold for the protein. Calcium binding to native Ī±LA is shown to help preserve the structure of the Ī²-domain of the protein limiting possibilities for disulfide bond shuffling. Hence, binding calcium plays an important role in both maintaining the native structure of Ī±LA and providing a mechanism for distinguishing between folded and misfolded species. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
A universal combinatorial design of antibody framework to graft distinct CDR sequences: A bioinformatics approach
Abstract Antibody (Ab) humanization is crucial to generate clinically relevant biologics from hybridoma-derived monoclonal antibodies (mAbs). In this study, we integrated antibody structural information from the Protein Data Bank with known back-to-mouse mutational data to build a universal consensus of framework positions (10 heavy and 7 light) critical for the preservation of the functional conformation of the Complimentarity Determining Region of antibodies. On the basis of FR consensus, we describe here a universal combinatorial library suitable for humanizing exogenous antibodies by CDR-grafting. The six CDRs of the murine anti-human EGFR Fab M225 were grafted onto a distinct (low FR sequence similarity to M225) human FR sequence that incorporates at the 17 FR consensus positions the permutations of the naturally observed amino acid diversities. Ten clones were selected from the combinatorial library expressing phage-displayed humanized M225 Fabs. Surprisingly, 2 of the 10 clones were found to bind EGFR with stronger affinity than M225. Cell-based assays demonstrated that the 10 selected clones retained epitope specificity by blocking EGFR phosphorylation and thus hindering cellular proliferation. Our results suggest that there is a universal and structurally rigid near-CDR set of FR positions that cooperatively support the binding conformation of CDRs. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Identification of the bile salt binding site on ipad from Shigella flexneri and the influence of ligand binding on IpaD structure
Abstract Type III secretion (TTS) is an essential virulence factor for Shigella flexneri , the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nano-machine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices Ī±3 and Ī±7, with concomitant movement in the orientation of helix Ī±7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC-bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain
Abstract Optimization of surface exposed chargeācharge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface chargeācharge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of chargeācharge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
H-bond network optimization in proteināprotein complexes: Are all-atom force field scores enough?
Abstract Structural prediction of proteināprotein complexes given the structures of the two interacting compounds in their unbound state is a key problem in biophysics. In addition to the problem of sampling of near-native orientations, one of the modeling main difficulties is to discriminate true from false positives. Here, we present a hierarchical protocol for docking refinement able to discriminate near native poses from a group of docking candidates. The main idea is to combine an efficient sampling of the full system hydrogen bond network and side chains, together with an all-atom force field and a surface generalized born implicit solvent. We tested our method on a set of twenty two complexes containing a near-native solution within the top 100 docking poses, obtaining a near native solution as the top pose in 70% of the cases. We show that all atom force fields optimized H-bond networks do improve significantly state of the art scoring functions. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
The N-terminal domains of SOCS proteins: A conserved region in the disordered N-termini of SOCS4 and 5
Abstract Suppressors of cytokine signaling (SOCS) proteins function as negative regulators of cytokine signaling and are involved in fine tuning the immune response. The structure and role of the SH2 domains and C-terminal SOCS box motifs of the SOCS proteins are well characterized, but the long N-terminal domains of SOCS4ā7 remain poorly understood. Here, we present bioinformatic analyses of the N-terminal domains of the mammalian SOCS proteins, which indicate that these domains of SOCS4, 5, 6, and 7 are largely disordered. We have also identified a conserved region of about 70 residues in the N-terminal domains of SOCS4 and 5 that is predicted to be more ordered than the surrounding sequence. The conservation of this region can be traced as far back as lower vertebrates. As conserved regions with increased structural propensity that are located within long disordered regions often contain molecular recognition motifs, we expressed the N-terminal conserved region of mouse SOCS4 for further analysis. This region, mSOCS486ā155 , has been characterized by circular dichroism and nuclear magnetic resonance spectroscopy, both of which indicate that it is predominantly unstructured in aqueous solution, although it becomes helical in the presence of trifluoroethanol. The high degree of sequence conservation of this region across different species and between SOCS4 and SOCS5 nonetheless implies that it has an important functional role, and presumably this region adopts a more ordered conformation in complex with its partners. The recombinant protein will be a valuable tool in identifying these partners and defining the structures of these complexes. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Ligand-induced formation of transient dimers of mammalian 12/15-lipoxygenase: A key to allosteric behavior of this class of enzymes?
Abstract Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomerādimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomerādimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S )-hydroxyoctadeca-9(Z ),11(E )-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
On the stoichiometry of Deinococcus radiodurans Dps-1 binding to duplex DNA
Abstract DNA protection during starvation (Dps) proteins, dodecameric assemblies of four-helix bundle subunits, contribute to protection against reactive oxygen species. Deinococcus radiodurans , which is characterized by resistance to DNA damaging agents, encodes two Dps homologs, of which Dps-1 binds DNA with high affinity. DNA binding requires N-terminal extensions preceding the four-helix bundle core. Composed of six Dps-1 dimers, each capable of DNA binding by N-terminal extensions interacting in consecutive DNA major grooves, dodecameric Dps-1 would be predicted to feature six DNA binding sites. Using electrophoretic mobility shift assays and intrinsic tryptophan fluorescence, we show that dodecameric Dps-1 binds 22-bp DNA with a stoichiometry of 1:6, consistent with the existence of six DNA binding sites. The stoichiometry of Dps-1 binding to 26-bp DNA is 1:4, suggesting that two Dps-1 dodecamers can simultaneously occupy opposite faces of this DNA. Mutagenesis of an arginine (Arg132) on the surface of Dps-1 leads to a reduction in DNA binding. Altogether, our data suggest that duplex DNA lies along the dimer interface, interacting with Arg132 and the N-terminal Ī±-helices, and they extend the hexagonal packing model for DpsāDNA assemblies by specifying the basis for occupancy of available DNA binding sites. Proteins 2011; Ā© 2012 Wiley Periodicals, Inc.
Computational protein design with explicit consideration of surface hydrophobic patches
Abstract De novo protein design requires the identification of amino-acid sequences that favor the target-folded conformation and are soluble in water. One strategy for promoting solubility is to disallow hydrophobic residues on the protein surface during design. However, naturally occurring proteins often have hydrophobic amino acids on their surface that contribute to protein stability via the partial burial of hydrophobic surface area or play a key role in the formation of proteināprotein interactions. A less restrictive approach for surface design that is used by the modeling program Rosetta is to parameterize the energy function so that the number of hydrophobic amino acids designed on the protein surface is similar to what is observed in naturally occurring monomeric proteins. Previous studies with Rosetta have shown that this limits surface hydrophobics to the naturally occurring frequency (ā¼ 28%), but that it does not prevent the formation of hydrophobic patches that are considerably larger than those observed in naturally occurring proteins. Here, we describe a new score term that explicitly detects and penalizes the formation of hydrophobic patches during computational protein design. With the new term, we are able to design protein surfaces that include hydrophobic amino acids at naturally occurring frequencies, but do not have large hydrophobic patches. By adjusting the strength of the new score term, the emphasis of surface redesigns can be switched between maintaining solubility and maximizing folding free energy. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Distance-dependent atomic knowledge-based force in protein fold recognition
Abstract We have recently introduced a novel model for discriminating the correctly folded proteins from well-designed decoy structures using mechanical interatomic forces. In the model, we considered a protein as a collection of springs and the force imposed to each atom was calculated by using the relation between the potential energy and the force. A mean force potential function is obtained from statistical contact preferences within the known protein structures. In this article, the interatomic forces are calculated by numerical derivation of the potential function. For assessing the knowledge-based force function we consider an optimal structure and define a score function on the 3D structure of a protein. We compare the force imposed to each atom of a protein with the corresponding atom in the optimum structure. Afterwards we assign larger scores to those atoms with the lower forces. The total score is the sum of partial scores of atoms. The optimal structure is assumed to be the one with the highest score in the dataset. Finally, several decoy sets are applied in order to evaluate the performance of our model. Proteins 2012. Ā© 2012 Wiley Periodicals, Inc.
Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G
Abstract Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNĪ³) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNĪ³ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K d values for hIFNĪ³ from 0.2 to 10 nM . Molecular modeling, computational docking onto hIFNĪ³, and in vitro competition for hIFNĪ³ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNĪ³, which differed from the site of human IFNĪ³ receptor 1 binding. Thus, these hIFNĪ³ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Kinetics and thermodynamics of gas diffusion in a NiFe hydrogenase
Abstract We have investigated O2 and H2 transport across a NiFe hydrogenase at the atomic scale by means of computational methods. The Wild Type protein has been compared with the V74Q mutant. Two distinct methodologies have been applied to study the gas access to the active site. Temperature locally enhanced sampling simulations have emphasized the importance of protein dynamics on gas diffusion. The O2 diffusion free energy profiles, obtained by umbrella sampling, are in agreement with the known kinetic data and show that in the V74Q mutant, the inhibition process is lowered from both a kinetic and a thermodynamic point of view. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Analysis on folding of misgurin using two-dimensional HP model
Abstract Misgurin is an antimicrobial peptide from the loach, while the hydrophobic-polar (HP) model is a way to study the folding conformations and native states in peptide and protein although several amino acids cannot be classified either hydrophobic or polar. Practically, the HP model requires extremely intensive computations, thus it has yet to be used widely. In this study, we use the two-dimensional HP model to analyze all possible folding conformations and native states of misgurin with conversion of natural amino acids according to the normalized amino acid hydrophobicity index as well as the shortest benchmark HP sequence. The results show that the conversion of misgurin into HP sequence with glycine as hydrophobic amino acid at pH 2 has 1212 folding conformations with the same native state of minimal energy ā6; the conversion of glycine as polar amino acid at pH 2 has 13,386 folding conformations with three native states of minimal energy ā5; the conversion of glycine as hydrophobic amino acid at pH 7 has 2538 folding conformations with three native states of minimal energy ā5; and the conversion of glycine as polar amino acid at pH 7 has 12,852 folding conformations with three native states of minimal energy ā4. Those native states can be ranked according to the normalized amino acid hydrophobicity index. The detailed discussions suggest two ways to modify misgurin. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
PDB-scale analysis of known and putative ligand-binding sites with structural sketches
Abstract Computational investigation of protein functions is one of the most urgent and demanding tasks in the field of structural bioinformatics. Exhaustive pairwise comparison of known and putative ligand-binding sites, across protein families and folds, is essential in elucidating the biological functions and evolutionary relationships of proteins. Given the vast amounts of data available now, existing 3D structural comparison methods are not adequate due to their computation time complexity. In this article, we propose a new bit string representation of binding sites called structural sketches, which is obtained by random projections of triplet descriptors. It allows us to use ultra-fast all-pair similarity search methods for strings with strictly controlled error rates. Exhaustive comparison of 1.2 million known and putative binding sites finished in ā¼30 h on a single core to yield 88 million similar binding site pairs. Careful investigation of 3.5 million pairs verified by TM-align revealed several notable analogous sites across distinct protein families or folds. In particular, we succeeded in finding highly plausible functions of several pockets via strong structural analogies. These results indicate that our method is a promising tool for functional annotation of binding sites derived from structural genomics projects. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Optimized grid-based proteināprotein docking as a global search tool followed by incorporating experimentally derivable restraints
Abstract Unbound protein docking, or the computational prediction of the structure of a protein complex from the structures of its separated components, is of importance but still challenging. A practical approach toward reliable results for unbound docking is to incorporate experimentally derived information with computation. To this end, truly systematic search of the global docking space is desirable. The fast Fourier transform (FFT) docking is a systematic search method with high computational efficiency. However, by using FFT to perform unbound docking, possible conformational changes upon binding must be treated implicitly. To better accommodate the implicit treatment of conformational flexibility, we develop a rational approach to optimize āsoftenedā parameters for FFT docking. In connection with the increased āsoftnessā of the parameters in this global search step, we use a revised rule to select candidate models from the search results. For complexes designated as of low and medium difficulty for unbound docking, these adaptations of the original FTDOCK program lead to substantial improvements of the global search results. Finally, we show that models resulted from FFT-based global search can be further filtered with restraints derivable from nuclear magnetic resonance (NMR) chemical shift perturbation or mutagenesis experiments, leading to a small set of models that can be feasibly refined and evaluated using computationally more expensive methods and that still include high-ranking near-native conformations. Proteins 2011. Ā© 2012 Wiley Periodicals, Inc.
Multiscale modelling of cellular actin filaments: From atomistic molecular to coarse grained dynamics
Abstract In this paper we present a computational multiscale model for the characterization of subcellular proteins. The model is encoded inside a simulation tool that builds coarse grained (CG) force fields from atomistic simulations. Equilibrium Molecular Dynamics simulations on an all-atom model of the actin filament are performed. Then, using the statistical distribution of the distances between pairs of selected groups of atoms at the output of the MD simulations, the force field is parameterized using the Boltzmann Inversion approach. This CG force field is further used to characterize the dynamics of the protein via Brownian Dynamics simulations. This combination of methods into a single computational tool-flow enables the simulation of actin filaments with length up to 400 nm, extending the time- and length-scales compared to state-of-the-art approaches. Moreover, the proposed multiscale modelling approach allows to investigate the relationship between atomistic structure and changes on the overall dynamics and mechanics of the filament and can be easily i ) extended to the characterization of other subcellular structures and ii ) used to investigate the cellular effects of molecular alterations due to pathological conditions. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Fold of the conserved DTC domain of deltex proteins
Abstract Human Deltex3-like (DTX3L) is a member of the Deltex family of proteins. Initially identified as a B-lymphoma and BAL-associated protein (BBAP), DTX3L is an E3 ligase that regulates sub cellular localization of its partner protein, BAL, by a dynamic nucleocytoplasmic trafficking mechanism. Unlike other members of the Deltex family of proteins, DTX3L lacks the highly basic N-terminal motif and the central proline rich motif present in other Deltex proteins, and instead contains other unique N-terminal domains. The C-terminal domains are however homologous with other members of the Deltex family of proteins; these include a RING domain and a previously unidentified C-terminal domain. In this study we report the high resolution crystal structure of this previously uncharacterized C-terminal domain of human DTX3L which we term the Deltex C-Terminal (DTC) domain. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Three-dimensional domain swapping in the protein structure space
Abstract Since the proposal of three-dimensional (3D) domain swapping, many 3D domain-swapped structures have been reported. However, compared with the vast protein structure space, it is still unclear whether 3D domain swapping is a general mechanism for protein assembly. Here, we investigated this possibility by constructing a dataset consisting of more than 500 domain-swapped structures. The domain-swapped structures were mapped into the protein structure space. We found that about 10% of protein folds and 5% of protein families contain domain-swapped structures. Comparing the domain-swapped structures in a family/superfamily, we found that proteins within a family/superfamily can swap in different ways. Interface analysis revealed that the hinge loops contributed more than half of the open interface in 70% of bona fide domain-swapped dimers, indicating that the hinge loops play an important role in stabilizing the domain-swapped conformations. Our study supports the suggestion that domain swapping is a general property of all proteins and will facilitate further understanding the mechanism of 3D domain swapping. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Simulations of substrate transport in the multidrug transporter EmrD
Abstract EmrD is a multidrug resistance (MDR) transporter from Escherichia coli, which is involved in the efflux of amphipathic compounds from the cytoplasm, and the first MDR member of the major facilitator superfamily (MFS) to be crystallized. Molecular dynamics simulation of EmrD in a phospholipid bilayer was used to characterize the conformational dynamics of the protein. Motions that support a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the EmrD central cavity were observed. Additionally, the translocation pathway of meta-chloro carbonylcyanide phenylhydrazone (CCCP) was probed using both standard and steered molecular dynamics simulation. In particular, interactions of a few specific residues with CCCP have been identified. Finally, a large motion of two residues, Val 45 and Leu 233, was observed with the passage of CCCP into the periplasmic space, placing a lower bound on the extent of opening required at this end of the protein for substrate transport. Overall, our simulations probe details of the transport pathway, motions of EmrD at an atomic level of detail, and offer new insights into the functioning of MDR transporters. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Insights on the acetylated NF-ĪŗB transcription factor complex with DNA from molecular dynamics simulations
Abstract The Nuclear Factor-ĪŗB (NF-ĪŗB) is a DNA sequence-specific regulator of many important biological processes, whose activity is modulated by enzymatic acetylation. In one of the best functionally characterized NF-ĪŗB complexes, the p50/p65 heterodimer, acetylation of K221 at p65 causes a decrease of DNA dissociation rate, whilst the acetylation of K122 and K123, also at p65, markedly decreases the binding affinity for DNA. By means of molecular dynamics simulations based on the X-ray structure of the p50/p65 complex with DNA, we provide here insights on the structural determinants of the acetylated complexes in aqueous solution. Lysine acetylation involves the loss of favourable electrostatic interactions between DNA and NF-ĪŗB, which is partially compensated by the reduction of the desolvation free-energy of the two binding partners. Acetylation at both positions K122 and K123 is associated with a decrease of the electrostatic potential at the p65/DNA interface, which is only partially counterbalanced by an increase of the local Na+ concentration. It induces the disruption of base-specific and non-specific interactions between DNA and NF-ĪŗB and it is consistent with the observed decrease of binding affinity. In contrast, acetylation at position K221 results in the loss of non-specific protein-DNA interactions, but the DNA recognition sites are not affected. In addition, the loss of protein-DNA interactions is likely to be counterbalanced by an increase of the configurational entropy of the complex, which provides, at a speculative level, a justification for the observed decrease of NF-ĪŗB/DNA dissociation rate. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
The nature of tryptophan radicals involved in the long-range electron transfer of Lignin Peroxidase (LiP) and LiP-like systems: Insights from QM/MM simulations
Abstract A catalytically active tryptophan radical has been demonstrated to be involved in the long-range electron transfer to the heme cofactor of Lignin Peroxidase (LiP) from Phanerochaete Chrysosporium although no direct detection by EPR spectroscopy of the tryptophan radical intermediate has been reported to date. An engineering-based approach has been used to manipulate the microenvironment of the redox-active tryptophan site in LiP and Coprinus cinereus Peroxidase (CiP), allowing the direct evidence of the tryptophan radical species. In light of the newly available EPR experimental data, we performed a QM/MM computational study in order to characterize the tryptophan radicals in the above protein matrices as well as in pristine LiP. The nature of the tryptophan radicals is discussed together with the analysis of their environment with the aim of understanding the different behaviour of pristine LiP in comparison to that of LiP and CiP variants. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
The ST pinch: A side chain-to-side chain hydrogen-bonded motif
Abstract The ST Pinch is a 12-membered hydrogen-bonded motif (Ser/Thr-Xaa-Ser/Thr) involving the side chain oxygen atoms of two Ser/Thr residues. We identified the ST Pinch in 104 proteins in a database containing high-resolution crystal structures. Conformational analysis of the ST Pinch in these proteins points to specific preferences for the Xaa residue and a high propensity of this residue to adopt positive Ļ angles. Our results suggest that this motif serves as a linker of secondary structural elements within proteins and is a new addition to the existing list of short hydrogen bond-stabilized motifs in proteins. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
A new and unexpected domain-domain interaction in the AraC protein
Abstract An interaction between the dimerization domains and DNA binding domains of the dimeric AraC protein has previously been shown to facilitate repression of the Escherichia coli araBAD operon by AraC in the absence of arabinose. A new interaction between the domains of AraC in the presence of arabinose is reported here, the regulatory consequences of which are unknown. Evidence for the interaction is the following: the dissociation rate of arabinose-bound AraC from half-site DNA is considerably faster than that of free DNA binding domain, and the affinity of the dimerization domains for arabinose is increased when half-site DNA is bound. In addition, an increase in the fluorescence intensity of tryptophan residues located in the arabinose-bound dimerization domain is observed upon binding of half-site DNA to the DNA binding domains. Direct physical evidence of the new domain-domain interaction is demonstrated by chemical crosslinking and NMR experiments. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
NMR-based insights into the conformational & interaction properties of Arkadia RING-H2 E3 Ub ligase
Abstract Arkadia (Rnf111), an E3 Ubiquitin (Ub) ligase, amplifies TGF-Ī² signaling responses by targeting for degradation the negative regulators Smad6/7 and the SnoN/Ski transcriptional repressors when they block the TGF-Ī² effectors Smad2/3. The E3 ligase activity of Arkadia depends on its C-terminal RING-H2 domain that constitutes the docking site for the E2 Ub-conjugating enzyme carrying the activated Ub. We determined the NMR solution-structure of Arkadia's RING-H2 domain and revealed a (Ī²)Ī²Ī²Ī± fold, fully consistent with the expected ācross-braceā mode of Zn(II)-ligation. Additionally, the interaction of the Arkadia RING-H2 domain with its E2 partner enzyme (UbcH5b) was examined through chemical shift perturbation. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish
Abstract The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from Zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Ć
), and adopts an overall globular shape. Two anti-parallel Ī²-sheets form a central Ī²-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Interactions between relay helix and Src homology 1 domain helix (SH1) drive the converter domain rotation during the recovery stroke of myosin II
Abstract Myosin motor protein exists in two alternative conformations, pre-recovery state M* and post-recovery state M**, upon ATP binding. The details of the M*-to-M** transition, known as the recovery stroke to reflect its role as the functional opposite of the force-generating power stroke, remain elusive. The defining feature of the post-recovery state is a kink in the relay helix, a key part of the protein involved in force generation. In this paper we determine the interactions that are responsible for the appearance of the kink. We design a series of computational models that contain three other segments, relay loop, converter domain and Src homology 1 domain helix (SH1), with which relay helix interacts, and determine their structure in accurate replica exchange molecular dynamics simulations in explicit solvent. By conducting an exhaustive combinatorial search among different models we find that: 1) the converter domain must be attached to the relay helix during the transition, so it does not interfere with other parts of the protein, 2) the structure of the relay helix is controlled by SH1 helix. The kink is strongly coupled to the position of SH1 helix. It arises as a result of direct interactions between SH1 and the relay helix and leads to a rotation of the C-terminal part of the relay helix which is subsequently transmitted to the converter domain. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Structural transitions and oligomerization along polyalanine fibril formation pathways from computer simulations
Abstract The results of a computer simulation study of the aggregation kinetics of a large system of model peptides with particular focus on the formation of intermediates are presented. Discontinuous molecular dynamic simulations were used in combination with our intermediate-resolution protein model, PRIME, to simulate the aggregation of a system of 192 polyalanine (KA14 K) peptides at a concentration of 5mM and reduced temperature of T* = 0.13 starting from a random configuration and ending in the assembly of a fibrillar structure. The population of various structures, including free monomers, beta sheets, amorphous aggregates, hybrid aggregates, and fibrils, and the transitions between the structures were tracked over the course of thirty independent simulations and averaged together. The aggregation pathway for this system starts with the association of free monomers into small amorphous aggregates that then grow to moderate size by incorporating other free monomers or merging with other small amorphous aggregates. These then rearrange into either small beta sheets or hybrid aggregates formed by association between unstructured chains and beta sheets, both of which grow in size by adding free monomer chains or other small aggregates, one at a time. Fibrillar structures are formed initially either by the stacking of beta sheets, rearrangement of hybrid aggregates or association between beta sheets and hybrid aggregates. They grow by the addition of beta sheets, hybrid aggregates and other small fibrillar structures. The rearrangement of amorphous aggregates into beta sheets is a critical and necessary step in the fibril formation pathway. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Structural features that predict real-value fluctuations of globular proteins
Abstract It is crucial to consider dynamics for understanding the biological function of proteins. We used a large number of molecular dynamics trajectories of non-homologous proteins as references and examined static structural features of proteins that are most relevant to fluctuations. We examined correlation of individual structural features with fluctuations and further investigated effective combinations of features for predicting the real-value of residue fluctuations using the support vector regression. It was found that some structural features have higher correlation than crystallographic B-factors with fluctuations observed in molecular dynamics trajectories. Moreover, support vector regression that uses combinations of static structural features showed accurate prediction of fluctuations with an average Pearson's correlation coefficient of 0.669 and a root mean square error of 1.04 Ć
. This correlation coefficient is higher than the one observed for the prediction by the Gaussian network model. An advantage of the developed method over the Gaussian network models is that the former predicts the real-value of fluctuation. The results help improve our understanding of relationships between protein structure and fluctuation. Furthermore, the developed method provides a convienient practial way to predict fluctuations of proteins using easily computed static structural features of proteins. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima
Abstract TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100Ā°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-Ī²-D-xylopyranoside monoacetates as substrates in a Ī²-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3 and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 Ć
and 2.5 Ć
resolution, respectively, revealing a classic Ī±/Ī²-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride (PMSF) and paraoxon were determined to 2.4 Ć
and 2.1 Ć
, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which this Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Crystal structure and biochemical properties of putrescine carbamoyltransferase from Enterococcus faecalis: Assembly, active site and allosteric regulation
Abstract Putrescine carbamoyltransferase (PTCase) catalyzes the conversion of carbamoylputrescine to putrescine and carbamoyl phosphate (CP), a substrate of carbamate kinase (CK). The crystal structure of PTCase has been determined and refined at 3.2 Ć
resolution. The trimeric molecular structure of PTCase is similar to other carbamoyltransferases, including the catalytic subunit of aspartate carbamoyltransferase (ATCase) and ornithine carbamoyltransferase (OTCase). However, in contrast to other trimeric carbamoyltransferases, PTCase binds both CP and putrescine with Hill coefficients at saturating concentrations of the other substrate of 1.53 Ā± 0.03 and 1.80 Ā± 0.06, respectively. PTCase also has a unique structural feature: a long C -terminal helix that interacts with the adjacent subunit to enhance intersubunit interactions in the molecular trimer. The C -terminal helix appears to be essential for both formation of the functional trimer and catalytic activity, since truncated PTCase without the C -terminal helix aggregates and has only 3% of native catalytic activity. The active sites of PTCase and OTCase are similar, with the exception of the 240ā²s loop. efPTCase lacks the proline-rich sequence found in knotted carbamoyltransferases and is unknotted. A Blast search of all available genomes indicates that 35 bacteria, most of which are Gram-positive, have an agcB gene encoding PTCase located near the genes that encode agmatine deiminase and carbamate kinase, consistent with the catabolic role of PTCase in the agmatine degradation pathway. The C -terminal helix identified in efPTCase is found in all other PTCases identified, suggesting that it is the signature feature of the PTCase family of enzymes. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics
Abstract Thioredoxins (TRXs) constitute attractive Ī±/Ī² scaffolds for investigating molecular recognition. The interaction between fragment TRX1-93 and peptide TRX94-108, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by DOSY-NMR; the pattern of secondary structure, as revealed by three-bond HNHĪ± coupling constants and secondary shifts for HĪ±/CO/CĪ±/CĪ²; native-like tertiary structural signatures revealed by near-UV CD spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear NOEs reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R2 values for residues 43-50 and 74-89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a K D
Molecular modeling of hair keratin/peptide complex: Using MM-PBSA calculations to describe experimental binding results
Abstract Molecular dynamics simulations of a keratin/peptide complex have been conducted to predict the binding affinity of four different peptides towards human hair. Free energy calculations on the peptides' interaction with the keratin model demonstrated that electrostatic interactions are believed to be the main driving force stabilizing the complex. The MM-PBSA methodology used for the free energy calculations demonstrated that the dielectric constant in the protein's interior plays a major role in the free energy calculations, and the only way to obtain accordance between the free energy calculations and the experimental binding results was to use the average dielectric constant. Ā© 2012 Wiley-Liss, Inc.
A synthetic hexapeptide designed to resemble a proteinaceous p-loop nest is shown to bind inorganic phosphate
Abstract The hexapeptide Ser-Gly-Ala-Gly-Lys-Thr has been synthesized and characterized. It was designed as a minimal soluble peptide that would be likely to have the phosphate-binding properties observed in the P-loops of proteins that bind the Ī²-phosphate of GTP or ATP. The Ī²-phosphate in such proteins is bound by a combination of the side chain Īµ-amino group of the lysine residue plus the concavity formed by successive main chain peptide NH groups called a nest, which is favored by the glycines. The hexapeptide is shown to bind HPO4 2- strongly at neutral pH. The affinities of the various ionized species of phosphate and hexapeptide are analyzed, showing that they increase with pH. It is likely the main chain NH groups of the hexapeptide bind phosphate in much the same way as the corresponding P-loop atoms bind the phosphate ligand in proteins. Most proteinaceous P-loops are situated at the N-termini of Ī±-helices and this observation has frequently been considered a key aspect of these binding sites. Such a hexapeptide in isolation seems unlikely to form an Ī±-helix, an expectation in accord with the CD spectra examined; this suggests that being at the N-terminus of an a-helix is not essential for phosphate binding. An unexpected finding about the hexapeptide-HPO4 2- complex is that the side chain Īµ-amino group of the lysine occurs in its deprotonated form, which appears to bind HPO4 2- via an Nā¦H-O-P hydrogen bond. Ā© 2012 Wiley-Liss, Inc.
The binding affinities of proteins interacting with the PDZ domain of PICK1
Abstract Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo1 (PDZ) proteinprotein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Do crystal structures obviate the need for theoretical models of GPCRs for structure based virtual screening
Abstract Recent highly expected structural characterizations of agonist-bound and antagonist-bound beta-2 adrenoreceptor (Ī²2AR) by X-ray crystallography have been widely regarded as critical advances to enable more effective structure-based discovery of GPCRs ligands. It appears that this very important development may have undermined many previous efforts to develop 3D theoretical models of GPCRs. To address this question directly we have compared several historical Ī²2AR models versus the inactive state and nanobody-stabilized active state of Ī²2AR crystal structures in terms of their structural similarity and effectiveness of use in virtual screening for Ī²2AR specific agonists and antagonists. Theoretical models, incluing both homology and de novo types, were collected from five different groups who have published extensively in the field of GPCRs modeling; all models were built before X-ray structures became available. In general, Ī²2AR theoretical models differ significantly from the crystal structure in terms of TMH definition and the global packing. Nevertheless, surprisingly, several models afforded hit rates resulting from virtual screening of large chemical library enriched by known Ī²2AR ligands that exceeded those using X-ray structures; the hit rates were particularly higher for agonists. Furthemore, the screening performance of models is associated with local structural quality such as the RMSDs for binding pocket residues and the ability to capture accurately most if not all critical protein/ligand interactions. These results suggest that carefully built models of GPCRs could capture critical chemical and structural features of the binding pocket thus may be even more useful for practical structure-based drug discovery than X-ray structures. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Towards a systems level view of the ECM and related proteins: A framework for the systematic definition and analysis of biological systems
Abstract Advances in high throughput 'omic technologies are starting to provide unprecedented insights into how components of biological systems are organized and interact. Key to exploiting these datasets is the definition of the components that comprise the system of interest. While a variety of knowledgebases exist that capture such information, a major challenge is determining how these resources may be best utilized. Here we present a systematic curation strategy to define a systems-level view of the human extracellular matrix (ECM) - a three-dimensional meshwork of proteins and polysaccharides that impart structure and mechanical stability to tissues. Employing our curation strategy we define a set of 357 proteins that represent core components of the ECM, together with an additional 524 genes that mediate related functional roles, and construct a map of their physical interactions. Topological properties help identify modules of functionally related proteins, including those involved in cell adhesion, bone formation and blood clotting. Due to its major role in cell adhesion, proliferation and morphogenesis, defects in the ECM have been implicated in cancer, atherosclerosis, asthma, fibrosis and arthritis. We use MeSH annotations to identify modules enriched for specific disease terms that aid to strengthen existing as well as predict novel gene-disease associations. Mapping expression and conservation data onto the network reveal modules evolved in parallel to convey tissue specific functionality on otherwise broadly expressed units. In addition to demonstrating an effective workflow for defining biological systems, this study crystallizes our current knowledge surrounding the organization of the ECM. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
The chaperone activity of Ī±-synuclein: Utilizing deletion mutants to map its interaction with target proteins
Abstract Ī±-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo , and under physiological conditions in vitro , Ī±-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. Ī±-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro . Ī±-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61ā140 [Ī±-syn(61ā140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments Ī±-syn(1ā60) and Ī±-syn(96ā140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar Ī±-synuclein had greater chaperone activity than nonfibrillar Ī±-synuclein. It is concluded that the amyloidogenic NAC region (residues 61ā95) contains the chaperone-binding site which is optimized for target protein binding as a result of its Ī²-sheet formation and/or ordered aggregation by Ī±-synuclein. On the other hand, the first 60 residues of Ī±-synuclein modulate the protein's chaperone-active site, while at the same time protecting Ī±-synuclein from fibrillation. On its own, however, this fragment [Ī±-syn(1ā60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of Ī±-synuclein in its chaperone activity have been delineated. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Large-scale conformational change in the mammalian prion protein massive conformation change in the prion protein
Abstract We employ all-atom structure-based models with a forcefield with multiple energetic basins for the C-terminal (residues 166-226) of the mammalian prion protein. One basin represents the known alpha-helical (Ī±H) structure while the other represents the same residues in a left-handed beta helical (LHBH) conformation. The LHBH structure has been proposed to help describe one class of in vitro grown fibrils, as well as possibly self-templating the conversion of normal cellular prion protein to the infectious form. Yet, it is unclear how the protein may make this global rearrangement. Our results demonstrate that the conformation changes are not strongly limited by large-scale geometry modification and that there may exist an overall preference for the LHBH conformation. Furthermore, our model presents novel intermediate trapping conformations with twisted LHBH structure. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Crystal structure and fluorescence studies reveal the role of helical dimeric interface of staphylococcal fabg1 in positive cooperativity for NADPH
Abstract Crystal structure of Staphylococcal Ī²-ketoacyl-ACP reductase 1 (SaFabG1) complexed with NADPH is determined at 2.5 Ć
resolution. The enzyme is essential in FAS-II pathway and utilizes NADPH to reduce Ī²-ketoacyl-ACP to (S )-Ī²-hydroxyacyl-ACP. Unlike the tetrameric FabGs, dimeric SaFabG1 shows positive homotropic cooperativity towards NADPH. Analysis of FabG:NADPH binary crystal structure endorses that NADPH interacts directly with the helices Ī±4 and Ī±5 those are present on a dimerization interface. A steady shift in tryptophan (of Ī±4 helix) emission peak upon steady increment of NADPH concentration reveals that the dimeric interface is formed by Ī±4-Ī±4ā² and Ī±5-Ī±5ā² helices. This dimeric interface imparts positive homotropic cooperativity towards NADPH. PEG, a substrate mimicking molecule is also found near the active site of the enzyme. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Thermodynamic basis of selectivity in guide-target-mismatched rna interference
Abstract Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2ā8 of the guide (āseed regionā); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed regionāa total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in targeting binding-free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also use this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNAā¢RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Engineering proteins with enhanced mechanical stability by force-specific sequence motifs
Abstract Use of atomic force microscopy (AFM) has recently led to a better understanding of the molecular mechanisms of the unfolding process by mechanical forces; however, the rational design of novel proteins with specific mechanical strength remains challenging. We have approached this problem from a new perspective that generates linear physicalāchemical properties (PCP) motifs from a limited AFM data set. Guided by our linear sequence analysis, we designed and analyzed four new mutants of the titin I1 domain with the goal of increasing the domain's mechanical strength. All four mutants could be cloned and expressed as soluble proteins. AFM data indicate that at least two of the mutants have increased molecular mechanical strength. This observation suggests that the PCP method is useful to graft sequences specific for high mechanical stability to weak proteins to increase their mechanical stability, and represents an additional tool in the design of novel proteins besides steered molecular dynamics calculations, coarse grained simulations, and Ļ-value analysis of the transition state. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Comparison of end-point continuum-solvation methods for the calculation of proteināligand binding free energies
Abstract We have compared the predictions of ligand-binding affinities from several methods based on end-point molecular dynamics simulations and continuum solvation, i.e. methods related to MM/PBSA (molecular mechanics combined with PoissonāBoltzmann and surface area solvation). Two continuum-solvation models were considered, viz. the PoissonāBoltzmann (PB) and generalised Born (GB) approaches. The non-electrostatic energies were also obtained in two different ways, viz. either from the sum of the bonded, van der Waals, non-polar solvation energies, and entropy terms (as in MM/PBSA), or from the scaled proteināligand van der Waals interaction energy (as in the linear interaction energy approach, LIE). Three different approaches to calculate electrostatic energies were tested, viz. the sum of electrostatic interaction energies and polar solvation energies, obtained either from a single simulation of the complex or from three independent simulations of the complex, the free protein, and the free ligand, or the linear-response approximation (LRA). Moreover, we investigated the effect of scaling the electrostatic interactions by an effective internal dielectric constant of the protein (Īµint ). All these methods were tested on the binding of seven biotin analogues to avidin and nine 3-amidinobenzyl-1H -indole-2-carboxamide inhibitors to factor Xa. For avidin, the best results were obtained with a combination of the LIE non-electrostatic energies with the MM+GB electrostatic energies from a single simulation, using Īµint = 4. For fXa, standard MM/GBSA, based on one simulation and using Īµint = 4ā10 gave the best result. The optimum internal dielectric constant seems to be slightly higher with PB than with GB solvation. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
A residue in helical conformation in the native state adopts a Ī²- strand conformation in the folding transition state despite its high and canonical Ī¦-value
Abstract Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Ī¦-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Ī¦-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Ī¦-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved Ī²-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Ī¦-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways, and demonstrates that even positions displaying high and canonical Ī¦- values may, nevertheless, adopt a non-native conformation in the transition state. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Signaling pathway of a photoactivable Rac1-GTPase in the early stages
Abstract In modern life- and medical-sciences major efforts are currently concentrated on creating arti-ficial photoenzymes, consisting of light-oxygen-voltage-sensitive (LOV) domains fused to a target enzyme. Such protein constructs possess great potential for controlling the cell metabolism as well as gene function upon light stimulus. This has recently been impressively demonstrated by de- signing a novel artificial fusion protein, connecting the AsLOV2-JĪ±-photosensor from Avena sativa with the Rac1-GTPase (AsLOV2-JĪ±-Rac1), and by using it, to control the motility of cancer cells from the HeLa-line. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their signaling pathway after photoexcitation is still in its infancy. Here, we show through computer simulations of the AsLOV2-JĪ±-Rac1-photoenzyme that the early processes after formation of the Cys450-FMN-adduct involve the breakage of a H- bond between the carbonyl oxygen FMN-C4=O and the amino group of Gln513, followed by a rotational re-orientation of its sidechain. This initial event is followed by successive events includ- ing Ī²-sheet tightening and transmission of torsional stress along the IĪ²-sheet, which leads to the disruption of the JĪ±-helix from the N-terminal end. Finally, this process triggers the detachment of the AsLOV2-JĪ±-photosensor from the Rac1-GTPase, ultimately enabling the activation of Rac1 via binding of the effector protein PAK1. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Signatures of the ATP binding pocket as a basis for structural classification of the serine/threonine protein kinases of gram-positive bacteria
Abstract Eukaryotic-like serine/threonine protein kinases (ESTPKs) are widely spread throughout the bacterial genomes. These enzymes can be potential targets of new anti-bacterial drugs useful for treatment of socially important diseases such as tuberculosis. In this study, ESTPKs of pathogenic, probiotic and antibiotic-producing gram-positive bacteria were classified according to the physico-chemical properties of amino acid residues in the ATP binding site of the enzyme. Nine residues were identified that line the surface of the adenine binding pocket, and ESTPKs were classified based on these signatures. Twenty groups were discovered, five of them containing >10 representatives. The two most abundant groups contained >150 protein kinases that belong to the various branches of the phylogenetic tree, whereas certain groups are genus- or even species-specific. Homology modeling of the typical representatives of each group revealed that the classification is reliable and the differences between the protein kinase ATP binding pockets predicted based on their signatures are apparent in their structure. The classification is expected to be useful for selection of targets for new anti-infective drugs. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin binding domains of utrophin and dystrophin
Abstract Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in-vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-Ī² aggregates, indicating a possible role for utrophin mutations in disease mechanisms. Proteins 2012. Ā© 2012 Wiley-Liss, Inc.
Conformational selection through electrostatics: Free energy simulations of gtp and gdp binding to archaeal initiation factor 2
Abstract Archaeal Initiation Factor 2 is a GTPase involved in protein biosynthesis. In its GTP-bound, āONā conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, āOFFā conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and their dependence on the conformational state, molecular dynamics free energy simulations were performed. The ON state specificity was predicted to be weak, with a GTP/GDP binding free energy difference of ā1 kcal/mol, favoring GTP. The OFF state specificity is larger, 4 kcal/mol, favoring GDP. The overall effects result from a competition among many interactions in several complexes. To interpret them, we use a simpler, dielectric continuum model. Several effects are robust with respect to the model details. Both nucleotides have a net negative charge, so that removing them from solvent into the binding pocket carries a desolvation penalty, which is large for the ON state, and strongly disfavors GTP binding compared to GDP. Short-range interactions between the additional GTP phosphate group and ionized sidechains in the binding pocket offset most, but not all of the desolvation penalty; more distant groups also contribute significantly, and the switch 1 loop only slightly. The desolvation penalty is lower for the more open, wetter OFF state, and the GTP/GDP difference much smaller. Short-range interactions in the binding pocket and with more distant groups again make a significant contribution. Overall, the simulations help explain how conformational selection is achieved with a single phosphate group. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Fast and accurate modeling of proteināprotein interactions by combining template-interface-based docking with flexible refinement
Abstract The similarity between folding and binding led us to posit the concept that the number of proteināprotein interface motifs in nature is limited, and interacting protein pairs can use similar interface architectures repeatedly, even if their global folds completely vary. Thus, known proteināprotein interface architectures can be used to model the complexes between two target proteins on the proteome scale, even if their global structures differ. This powerful concept is combined with a flexible refinement and global energy assessment tool. The accuracy of the method is highly dependent on the structural diversity of the interface architectures in the template dataset. Here, we validate this knowledge-based combinatorial method on the Docking Benchmark and show that it efficiently finds high-quality models for benchmark complexes and their binding regions even in the absence of template interfaces having sequence similarity to the targets. Compared to āclassicalā docking, it is computationally faster; as the number of target proteins increases, the difference becomes more dramatic. Further, it is able to distinguish binders from nonbinders. These features allow performing large-scale network modeling. The results on an independent target set (proteins in the p53 molecular interaction map) show that current method can be used to predict whether a given protein pair interacts. Overall, while constrained by the diversity of the template set, this approach efficiently produces high-quality models of proteināprotein complexes. We expect that with the growing number of known interface architectures, this type of knowledge-based methods will be increasingly used by the broad proteomics community. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Exploring c-Met kinase flexibility by sampling and clustering its conformational space
Abstract It is now widely recognized that the flexibility of both partners has to be considered in molecular docking studies. However, the question how to handle the best the huge computational complexity of exploring the protein binding site landscape is still a matter of debate. Here we investigate the flexibility of c-Met kinase as a test case for comparing several simulation methods. The c-Met kinase catalytic site is an interesting target for anticancer drug design. In particular, it harbors an unusual plasticity compared with other kinases ATP binding sites. Exploiting this feature may eventually lead to the discovery of new anticancer agents with exquisite specificity. We present in this article an extensive investigation of c-Met kinase conformational space using large-scale computational simulations in order to extend the knowledge already gathered from available X-ray structures. In the process, we compare the relevance of different strategies for modeling and injecting receptor flexibility information into early stage in silico structure-based drug discovery pipeline. The results presented here are currently being exploited in on-going virtual screening investigations on c-Met. Proteins 2012;. Ā© 2011 Wiley Periodicals, Inc.
Examining the promiscuous phosphatase activity of Pseudomonas aeruginosa arylsulfatase: A comparison to analogous phosphatases
Abstract Pseudomonas aeruginosa arylsulfatase (PAS) is a bacterial sulfatase capable of hydrolyzing a range of sulfate esters. Recently, it has been demonstrated to also show very high proficiency for phosphate ester hydrolysis. Such proficient catalytic promiscuity is significant, as promiscuity has been suggested to play an important role in enzyme evolution. Additionally, a comparative study of the hydrolyses of the p -nitrophenyl phosphate and sulfate monoesters in aqueous solution has demonstrated that despite superficial similarities, the two reactions proceed through markedly different transition states with very different solvation effects, indicating that the requirements for the efficient catalysis of the two reactions by an enzyme will also be very different (and yet they are both catalyzed by the same active site). This work explores the promiscuous phosphomonoesterase activity of PAS. Specifically, we have investigated the identity of the most likely base for the initial activation of the unusual formylglycine hydrate nucleophile (which is common to many sulfatases), and demonstrate that a concerted substrate-as-base mechanism is fully consistent with the experimentally observed data. This is very similar to other related systems, and suggests that, as far as the phosphomonoesterase activity of PAS is concerned, the sulfatase behaves like a āclassicalā phosphatase, despite the fact that such a mechanism is unlikely to be available to the native substrate (based on pK a considerations and studies of model systems). Understanding such catalytic versatility can be used to design novel artificial enzymes that are far more proficient than the current generation of designer enzymes. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics
Abstract Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb 6PGL) with (holo -) and without (apo -) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of proteinātarget interactions. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison
Abstract Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Proteins 2012;. Ā© 2011 Wiley Periodicals, Inc.
Dynamics alignment: Comparison of protein dynamics in the scop database
Abstract A novel methodology for comparison of protein dynamics is presented. Protein dynamics is calculated using the Gaussian network model and the modes of motion are globally aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. The alignment is fast and can be used to analyze large sets of proteins. The methodology is applied to the four major classes of the SCOP database: āall alpha proteins,ā āall beta proteins,ā āalpha and beta proteins,ā and āalpha/beta proteinsā. We show that different domains may have similar global dynamics. In addition, we report that the dynamics of āall alpha proteinsā domains are less specific to structural variations within a given fold or superfamily compared with the other classes. We report that domain pairs with the most similar and the least similar global dynamics tend to be of similar length. The significance of the methodology is that it suggests a new and efficient way of mapping between the global structural features of protein families/subfamilies and their encoded dynamics. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Structural thermal adaptation of Ī²-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii
Abstract Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4Ā°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three Ī²-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4Ā°C. In comparison to the Ī²-tubulin from E. crassus , a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii Ī²-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic Ī²-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties. Proteins 2012;. Ā© 2011 Wiley Periodicals, Inc.
Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function
Abstract The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P2 ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-Pā¢F-6-P), in a transition stateāanalogous complex (PFKFB3ā¢AlF4 ), and in a complex with pyrophosphate (PFKFB3ā¢PPi ) at resolutions of 2.45, 2.2, and 2.3 Ć
, respectively. Trapping the PFKFB3-Pā¢F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3ā¢AlF4 and PFKFB3ā¢PPi complexes were obtained by soaking. The PFKFB3ā¢AlF4 and PFKFB3ā¢PPi complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ā¼5.1 Ć
, and the pivotal point 2-P is on the same line, suggesting an āin-lineā transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent Ni , implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P2 ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Altered nuclear cofactor switching in retinoic-resistant variants of the PML-RARĪ± oncoprotein of acute promyelocytic leukemia
Abstract Acute promyelocytic leukemia (APL) results from a reciprocal translocation that fuses the gene for the PML tumor suppressor to that encoding the retinoic acid receptor alpha (RARĪ±). The resulting PML-RARĪ± oncogene product interferes with multiple regulatory pathways associated with myeloid differentiation, including normal PML and RARĪ± functions. The standard treatment for APL includes anthracycline-based chemotherapeutic agents plus the RARĪ± agonist all-trans retinoic acid (ATRA). Relapse, which is often accompanied by ATRA resistance, occurs in an appreciable frequency of treated patients. One potential mechanism suggested by model experiments featuring the selection of ATRA-resistant APL cell lines involves ATRA-resistant versions of the PML-RARĪ± oncogene, where the relevant mutations localize to the RARĪ± ligand-binding domain (LBD). Such mutations may act by compromising agonist binding, but other mechanisms are possible. Here, we studied the molecular consequence of ATRA resistance by use of circular dichroism, protease resistance, and fluorescence anisotropy assays employing peptides derived from the NCOR nuclear corepressor and the ACTR nuclear coactivator. The consequences of the mutations on global structure and cofactor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated cofactor switching and increased protease resistance represent features of the LBDs of ATRA-resistant PML-RARĪ±, and these properties may be recapitulated in the full-length oncoproteins. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Validating the vitality strategy for fighting drug resistance
Abstract The current challenge in designing effective drugs against HIV-1 is to find novel candidates with high potency, but with a lower susceptibility to mutations associated with drug resistance. Trying to address this challenge, we developed in our previous study (Ishikita and Warshel, Angew Chem Int Ed Engl 2008; 47:697ā700) a novel computational strategy for fighting drug resistance by predicting the likely moves of the virus through constraints on binding and catalysis. This has been based on calculating the ratio between the vitality values ((Ki k cat /K M )mutant /(Ki k cat /K M )wild-type ) and using it as a guide for predicting the moves of the virus. The corresponding calculations of the binding affinity, K i k cat /K M , by evaluating the transition state (TS) binding free energies using the PDLD/S-LRA/Ī² method. Here we provide an extensive validation of our strategy by calculating the vitality of six existing clinical and experimental drug candidates. It is found that the computationally determined vitalities correlate reasonably well with those derived from the corresponding experimental data. This indicates that the calculated vitality may be used to identify mutations that would be most effective for the survival of the virus. Thus, it should be possible to use our approach in screening for mutations that would provide the most effective resistance to any proposed antiviral drug. This ability should be very useful in guiding the design of drug molecules that will lead to the slowest resistance. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Molecular insight into substrate recognition by human cytosolic sialidase NEU2
Abstract Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide Ī±(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide Ī±(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
The effects of rigid motions on elastic network model force constants
Abstract Elastic network models provide an efficient way to quickly calculate protein global dynamics from experimentally determined structures. The model's single parameter, its force constant, determines the physical extent of equilibrium fluctuations. The values of force constants can be calculated by fitting to experimental data, but the results depend on the type of experimental data used. Here, we investigate the differences between calculated values of force constants and data from NMR and X-ray structures. We find that X-ray B factors carry the signature of rigid-body motions, to the extent that B factors can be almost entirely accounted for by rigid motions alone. When fitting to more refined anisotropic temperature factors, the contributions of rigid motions are significantly reduced, indicating that the large contribution of rigid motions to B factors is a result of over-fitting. No correlation is found between force constants fit to NMR data and those fit to X-ray data, possibly due to the inability of NMR data to accurately capture protein dynamics. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Role of amino acid hydrophobicity, aromaticity, and molecular volume on IAPP(20ā29) amyloid self-assembly
Abstract Aromatic amino acids strongly promote cross-Ī² amyloid formation; whether the amyloidogenicity of aromatic residues is due to high hydrophobicity and Ī²-sheet propensity or formation of stabilizing ĻāĻ interactions has been debated. To clarify the role of aromatic residues on amyloid formation, the islet amyloid polypeptide 20ā29 fragment [IAPP(20ā29)], which contains a single aromatic residue (Phe 23), was adopted as a model. The side chain of residue 23 does not self-associate in cross-Ī² fibrils of IAPP(20ā29) (Nielsen et al., Angew Chem Int Ed 2009;48:2118ā2121), allowing investigation of the amyloidogenicity of aromatic amino acids in a context where direct ĻāĻ interactions do not occur. We prepared variants of IAPP(20ā29) in which Tyr, Leu, Phe, pentafluorophenylalanine (F5-Phe), Trp, cyclohexylalanine (Cha), Ī±-naphthylalanine (1-Nap), or Ī²-naphthylalanine (2-Nap) (in order of increasing peptide hydrophobicity) were incorporated at position 23 (SNNXGAILSS-NH2), and the kinetic and thermodynamic effects of these mutations on cross-Ī² self-assembly were assessed. The Tyr, Leu, and Trp 23 variants failed to readily self-assemble at concentrations up to 1.5 mM , while the Cha 23 mutant fibrillized with attenuated kinetics and similar thermodynamic stability relative to the wild-type Phe 23 peptide. Conversely, the F5-Phe, 1-Nap, and 2-Nap 23 variants self-assembled at enhanced rates, forming fibrils with greater thermodynamic stability than the wild-type peptide. These results indicate that the high amyloidogenicity of aromatic amino acids is a function of hydrophobicity, Ī²-sheet propensity, and planar geometry and not the ability to form stabilizing or directing ĻāĻ bonds. Proteins 2012;. Ā© 2011 Wiley Periodicals, Inc.
Automatch: Target-binding protein design and enzyme design by automatic pinpointing potential active sites in available protein scaffolds
Abstract Proteins perform their functions mainly via active sites, whereas other parts of the proteins comprise the scaffolds, which support the active sites. One strategy for protein functional design is transplanting active sites, such as catalytic sites for enzyme or binding hot spots for proteināprotein interactions, onto a new scaffold. AutoMatch is a new program designed for efficiently elucidating suitable scaffolds and potential sites on the scaffolds. Backrub motions are used to treat backbone flexibility during the design process. A step-by-step checking strategy and cluster-representation examination strategy were developed to solve the large combinatorial problem for the matching of active-site conformations. In addition, a grid-based binding energy scoring method was used to filter the solutions. An enzyme design benchmark and a proteināprotein interaction design benchmark were built to test the algorithm. AutoMatch could identify the hot spots in the nonbinding protein and rank them within the top five results for 8 of 10 target-binding protein design cases. In addition, among the 15 enzymes tested, AutoMatch can identify the catalytic active sites in the apoprotein and rank them within the top five results for 13 cases. AutoMatch was also tested for screening scaffold library in designing binding proteins targeting influenza hemagglutinin, HIV gp120, and epidermal growth factor receptor kinase, respectively. AutoMatch, and the two test sets, ActApo and ActFree, are available for noncommercial applications at http://mdl.ipc.pku.edu.cn/cgi-bin/down.cgi . Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
Conformational dynamics of capping protein and interaction partners: Simulation studies
Abstract Capping protein (CP) is important for the regulation of actin polymerization. CP binds to the barbed end of the actin filament and prevents actin polymerization. This interaction is modulated through competitive binding by regulatory proteins such as myotrophin (V-1) and the capping protein interacting (CPI) motif from CARMIL. The binding site of myotrophin overlaps with the region of CP that binds to the barbed end of actin filament, whereas CPI binds at a distant site. The binding of CPI to the myotrophin-CP complex dissociates myotrophin from CP. Detailed multicopy molecular dynamics simulations suggest that the binding of CPI shifts the conformational equilibria of CP away from states that favor myotrophin binding. This shift is underpinned by allosteric effects where CPI inhibits CP through suppression of flexibility and disruption of concerted motions that appear to mediate myotrophin binding. Accompanying these effects are changes in electrostatic interactions, notably those involving residue K142Ī², which appears to play a critical role in regulating flexibility. In addition, accessibility of the site on CP for binding the key hydrophobic residue W8 of myotrophin is modulated by CPI. These results provide insights into the modulation of CP by CPI and myotrophin and indicate the mechanism by which CPI drives the dissociation of the myotrophin-CP complex. Proteins 2012;. Ā© 2011 Wiley Periodicals, Inc.
Determination of the dipole moments of RNAse SA wild type and a basic mutant
Abstract In this study, we report the effects of acidic to basic residue point mutations (5K) on the dipole moment of RNAse SA at different pHs. Dipole moments were determined by measuring solution capacitance of the wild type (WT) and the 5K mutant with an impedance analyzer. The dipole moments were then (1) compared with theoretically calculated dipole moments, (2) analyzed to determine the effect of the point mutations, and (3) analyzed for their contribution to overall proteināprotein interactions (PPI) in solution as quantitated by experimentally derived second virial coefficients. We determined that experimental and calculated dipoles were in reasonable agreement. Differences are likely due to local motions of residue side chains, which are not accounted for by the calculated dipole. We observed that the proteins' dipole moments increase as the pH is shifted further from their isoelectric points and that the wild-type dipole moments were greater than those of the 5K. This is likely due to an increase in the proportion of one charge (either negative or positive) relative to the other. A greater charge disparity corresponded to a larger dipole moment. Finally, the larger dipole moments of the WT resulted in greater attractive overall PPI for that protein as compared to the 5K. Proteins 2011; Ā© 2011 Wiley Periodicals, Inc.
Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis
Abstract The catalytic adenyl cyclase (AC) domain of the protein CyaA from Bordetella pertussis is activated by interaction with the C terminal lobe of calmodulin (C-CaM). The AC/C-CaM complex displays an elongated shape, but hydrodynamics measurements on the isolated AC domain allowed to characterize the shape of the protein as spherical. Here, we study by molecular dynamics simulations the complexes between AC and the apo and Ca2+ -loaded C-CaM, as well as the isolated AC, to characterize the features of AC conformational variability and of AC/C-CaM interaction. The removal of calcium ions from C-CaM increases the AC flexibility, but the removal of C-CaM induces a dramatic drift of the AC conformation. Isolated AC conformations show a general tendency to become less elongated, as the two protein extremities (regions SA and CB) tend to get closer. An analysis of the energetic influences between the C-CaM and the AC regions shows a simple influence scheme, in agreement with the high affinity of AC to CaM. In this scheme, a single influence is observed from C-CaM to the region CA of the AC domain. This influence is correlated to the presence of hydrogen bonds involving residues from C-CaM, and from regions CA, C-terminal tail, and catalytic loop of AC. This study reveals a C-CaM/AC interaction picture where C-CaM stabilizes AC by a steric hindrance on the conformational drift of SA, whereas the Ca2+ ions allow further stabilization by the establishment of a hydrogen bond network extending from C-CaM to the AC catalytic loop. Proteins 2011;. Ā© 2011 Wiley Periodicals, Inc.
Crystal structure of the Kar3-like kinesin motor domain from the filamentous fungus Ashbya gossypii
Abstract Kar3 kinesins are microtubule (MT) minus-end-directed motors with pleiotropic functions in mitotic spindle formation and nuclear movement in budding and fission yeasts. A Kar3-like kinesin is also expressed by the filamentous fungus Ashbya gossypi, which exhibits different nuclear movement challenges from its yeast relatives. Presented here is a 2.35 Ć
crystal structure and enzymatic analysis of the Ag Kar3 motor domain (Ag Kar3MD). Compared to the previously published Saccharomyces cerevisiae Kar3MD structure (Sc Kar3MD), Ag Kar3MD displays differences in the conformation of some of its nucleotide-binding motifs and peripheral elements. Unlike Sc Kar3MD, the salt bridge between Switch I and Switch II in Ag Kar3MD is broken. Most of the Switch I, and the adjoining region of helix Ī±3, are also disordered instead of bending into the active site cleft as is observed in Sc Kar3MD. These aspects of Ag Kar3MD are highly reminiscent of the Sc Kar3 R598A mutant that disrupts the Switch IāSwitch II salt bridge and impairs MT-stimulated ATPase activity of the motor. Subtle differences in the disposition of secondary structure elements in the small lobe (Ī²1a, Ī²1b, and Ī²1c) at the edge of the MD are also apparent even though it contains approximately the same number of residues as Sc Kar3. These differences may reflect the unique enzymatic properties we measured for this motor, which include a lower MT-stimulated ATPase rate relative to Sc Kar3, or they could relate to its interactions with different regulatory companion proteins than its budding yeast counterpart. Proteins 2011;. Ā© 2011 Wiley Periodicals, Inc.
How do thermophilic proteins resist aggregation?
Abstract Aggregation is an ancient threat that must be overcome by proteins from all organisms to maintain their native functional states. This is essential for the maintenance of metabolic flux and viability of their cellular machineries. Here, we compare the aggregation-resistance strategies adapted by the thermophilic proteins and their mesophilic homologs using a dataset of 373 protein families. Like their mesophilic homologs, the thermophilic protein sequences also contain potential aggregation prone regions (APRs), capable of forming cross-Ī² motif and amyloid-like fibrils. Tetrapeptide and hexapeptide amyloid-like fibril forming sequence patterns and experimentally proven amyloid-like fibril forming peptide sequences were also detected in the thermophilic proteins. Both the thermophilic and the mesophilic proteins use similar strategies to resist aggregation. However, the thermophilic proteins show superior utilization of these strategies. The thermophilic protein monomers show greater ability to āstow awayā the APRs in the hydrophobic cores to protect them from solvent exposure. The thermophilic proteins are also better at gatekeeping the APRs by surrounding them with charged residues (Asp, Glu, Lys, and Arg) and Pro to a greater extent. While thermophilic and mesophilic proteins in our dataset are highly homologous and show strong overall sequence conservation, the APRs are not conserved between the homologs. These findings indicate that evolution is working to avoid amyloidogenic regions in proteins. Our results are also consistent with the observation that thermophilic cells often accumulate small molecule osmolytes capable of stabilizing their proteins and other macromolecules. This study has important implications for rational design and formulation of therapeutic proteins and antibodies. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
A comprehensive library of blocked dipeptides reveals intrinsic backbone conformational propensities of unfolded proteins
Abstract Despite prolonged scientific efforts to elucidate the intrinsic peptide backbone preferences of amino-acids based on understanding of intermolecular forces, many open questions remain, particularly concerning neighboring peptide interaction effects on the backbone conformational distribution of short peptides and unfolded proteins. Here, we show that spectroscopic studies of a complete library of 400 dipeptides reveal that, irrespective of side-chain properties, the backbone conformation distribution is narrow and they adopt polyproline II and Ī²-strand, indicating the importance of backbone peptide solvation and electronic effects. By directly comparing the dipeptide circular dichroism and NMR results with those of unfolded proteins, the comprehensive dipeptides form a complete set of structural motifs of unfolded proteins. We thus anticipate that the present dipeptide library with spectroscopic data can serve as a useful database for understanding the nature of unfolded protein structures and for further refinements of molecular mechanical parameters. Proteins 2011; Ā© 2011 Wiley Periodicals, Inc.
Asparagine and glutamine differ in their propensities to form specific side chain-backbone hydrogen bonded motifs in proteins
Abstract Short range side chain-backbone hydrogen bonded motifs involving Asn and Gln residues have been identified from a data set of 1370 protein crystal structures (resolution ā¤ 1.5 Ć
). Hydrogen bonds involving residues i ā 5 to i + 5 have been considered. Out of 12,901 Asn residues, 3403 residues (26.4%) participate in such interactions, while out of 10,934 Gln residues, 1780 Gln residues (16.3%) are involved in these motifs. Hydrogen bonded ring sizes (Cn n is the number of atoms involved), directionality and internal torsion angles are used to classify motifs. The occurrence of the various motifs in the contexts of protein structure is illustrated. Distinct differences are established between the nature of motifs formed by Asn and Gln residues. For Asn, the most highly populated motifs are the C10 (COĪ“ i ā¦NHi + 213 (COĪ“ i ā¦NHi + 317 (NĪ“ Hi ā¦COi ā 416 (COĪµ i ā¦NHi ā 312 (NĪµ Hi ā¦COi ā 215 (NĪµ Hi i ā 318 (NĪµ Hi ā¦COi ā 418 motif being analogous to the Asn C17 structure. Specific conformational types are established for the Asn containing motifs, which mimic backbone Ī²-turns and Ī±-turns. Histidine residues are shown to serve as a mimic for Asn residues in side chain-backbone hydrogen bonded ring motifs. Illustrative examples from protein structures are considered. Proteins 2012; Ā© 2011 Wiley Periodicals, Inc.
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Open and closed conformations reveal induced fit movements in butyrate kinase 2 activation
Abstract Butyrate kinase catalyzes the phosphorylation of butyrate, or converts butyryl phosphate to butyrate in the reverse phosphorylation reaction in the last step of butyrate fermentation. Butyrate fermentation is one of several major fermentations and the fermentation product butyrate has been implicated in protection from colon cancer. Butyrate kinase belongs to the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases, of which all the members share a conserved two-domain structural fold. Here we report the 3.0 Ć
crystal structure of octameric butyrate kinase 2 (Buk2) from Thermotoga maritima in which six monomers are in the open conformation and the other two monomers are in the citrate-bound, closed form. The bound citrate induces an 18-degree rotation of the N-terminal domain relative to the C-terminal domain, corresponding to movements of the polypeptide backbone of 8.5 Ć
. The N- and C-terminal domains bend around two hinges, which leads to the rigid-body movements of the two domains and a dramatic closing of the active site cleft. The two monomers in the closed form afford an observation of the active site in a catalytic conformation. The substrate docking and the simulation of the open to closed conformational changes illustrate an induced fit mechanism. The citrate-induced conformational changes may help understand the movements of Buk2 as a result of butyrate/isobutyrate binding, and further structural comparisons and discussions provide overall understanding of the structural biology of the ASKHA superfamily. Proteins 2010. Ā© 2009 Wiley-Liss, Inc.
![[BOND]](http://onlinelibrarystatic.wiley.com/undisplayable_characters/00f8ff.gif)
P bond, breaking the 2O