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BMC Structural Biology - Editor: BioMed Central
BMC Structural Biology is an open access journal publishing original peer-reviewed research articles in investigations into the structure of biological macromolecules, including solving structures, structural and functional analyses, and computational modeling.
Small-angle scattering is becoming an increasingly popular tool for the study of bio-molecular structures in solution. The large number of publications with 3D-structural models generated from small-angle solution scattering data has led to a growing consensus for the need to establish a standard reporting framework for their publication. The International Union of Crystallography recently established a set of guidelines for the necessary information required for the publication of such structural models. Here we describe the rationale for these guidelines and the importance of standardising the way in which small-angle scattering data from bio-molecules and associated structural interpretations are reported.
Background:
Tumor necrosis factors, TNF and lymphotoxin-alpha (LT), are cytokines that bind to two receptors, TNFR1 and TNFR2 (TNF-receptor 1 and 2) to trigger their signaling cascades. The exact mechanism of ligand-induced receptor activation is still unclear. It is generally assumed that three receptors bind to the homotrimeric ligand to trigger a signaling event. Recent evidence, though, has raised doubts if the ligand:receptor stoichiometry should indeed be 3:3 for ligand-induced cellular response. We used molecular dynamics simulations, elastic network models, as well as MM/PBSA to analyze this question.
Results:
Applying MM/PBSA methodology to different stoichiometric complexes of human LT-(TNFR1)n=1,2,3 the free energy of binding in these complexes has been estimated by single-trajectory and separate-trajectory methods. Simulation studies rationalized the favorable binding energy in the LT-(TNFR1)1 complex, as evaluated from single-trajectory analysis to be an outcome of the interaction of cysteine-rich domain 4 (CRD4) and the ligand. Elastic network models (ENMs) help to associate the difference in the global fluctuation of the receptors in these complexes. Functionally relevant transformation associated with these complexes reveal the difference in the dynamics of the receptor when in unbound form and in complex with LT.
Conclusions:
MM/PBSA predicts complexes with a ligand-receptor molar ratio of 3:1 and 3:2 to be energetically favorable. The high affinity associated with LT-(TNFR1)1 is due to the interaction between the CRD4 domain with LT. The global dynamics ascertained from ENMs have highlighted the differential dynamics of the receptor in different states.
Background:
Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (2) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor 2 uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the N-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite.
Results:
Inhibitor 2 {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 A X-ray data. The benzene ring of the inhibitor tilted 8.9degrees compared to the previous compound (1), and the number of contacts, including hydrogen bonds, increased. However, the IC50 for compound 2 remained in the low micromolar range, likely because one propyl group was disordered.In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit.
Conclusions:
We designed inhibitor 2 with an extended substituent at C4 corresponding to C6 of sialic acid to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 A showed that we partially succeeded in that the ring in 2 is tilted relative to 1 and the number of contacts increased, but one hydrophobic branch makes no contacts, perhaps explaining why the IC50 did not decrease. Future design efforts will include branches of unequal length so that both branches may be accommodated in the C6-subsite without conformational disorder.The high-mannose glycan attached to Asn200 makes several inter-subunit contacts and appears to stabilize the tetramer.
Background:
Most signalling and regulatory proteins participate in transient protein-protein interactions during biological processes. They usually serve as key regulators of various cellular processes and are often stable in both protein-bound and unbound forms. Availability of high-resolution structures of their unbound and bound forms provides an opportunity to understand the molecular mechanisms involved. In this work, we have addressed the question "What is the nature, extent, location and functional significance of structural changes which are associated with formation of protein-protein complexes?"
Results:
A database of 76 non-redundant sets of high resolution 3-D structures of protein-protein complexes, representing diverse functions, and corresponding unbound forms, has been used in this analysis. Structural changes associated with protein-protein complexation have been investigated using structural measures and Protein Blocks description. Our study highlights that significant structural rearrangement occurs on binding at the interface as well as at regions away from the interface to form a highly specific, stable and functional complex. Notably, predominantly unaltered interfaces interact mainly with interfaces undergoing substantial structural alterations, revealing the presence of at least one structural regulatory component in every complex.Interestingly, about one-half of the number of complexes, comprising largely of signalling proteins, show substantial localized structural change at surfaces away from the interface. Normal mode analysis and available information on functions on some of these complexes suggests that many of these changes are allosteric. This change is largely manifest in the proteins whose interfaces are altered upon binding, implicating structural change as the possible trigger of allosteric effect. Although large-scale studies of allostery induced by small-molecule effectors are available in literature, this is, to our knowledge, the first study indicating the prevalence of allostery induced by protein effectors.
Conclusions:
The enrichment of allosteric sites in signalling proteins, whose mutations commonly lead to diseases such as cancer, provides support for the usage of allosteric modulators in combating these diseases.
Background:
Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance) of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities.
Results:
We showed that radial distribution functions (RDFs), which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments.
Conclusions:
Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites.
In our article (Sirim et al. BMC Struct Biol 2010, 10: 34) the cytochrome P450 monooxygenase P450cam was referred to as CYP101D. In the latest release 3.0 of our cytochrome P450 database CYPED (www.CYPED.uni-stuttgart.de) P450cam was reassigned to family CYP101A and referred to as CYP101A1.
Background:
There exist > 78,000 proteins and/or nucleic acids structures that were determined experimentally. Only a small portion of these structures corresponds to those of protein complexes. While homology modeling is able to exploit knowledge-based potentials of side-chain rotomers and backbone motifs to infer structures for new proteins, no such general method exists to extend our understanding of protein interaction motifs to novel protein complexes.
Results:
We use a Motif Binding Geometries (MBG) approach, to infer the structure of a protein complex from the database of complexes of homologous proteins taken from other contexts (such as the helix-turn-helix motif binding double stranded DNA), and demonstrate its utility on one of the more important regulatory complexes in biology, that of the RNA polymerase initiating transcription under conditions of phosphate starvation. The modeled PhoB/RNAP/?-factor/DNA complex is stereo-chemically reasonable, has sufficient interfacial Solvent Excluded Surface Areas (SESAs) to provide adequate binding strength, is physically meaningful for transcription regulation, and is consistent with a variety of known experimental constraints.
Conclusions:
Based on a straightforward and easy to comprehend concept, "proteins and protein domains that fold similarly could interact similarly", a structural model of the PhoB dimer in the transcription initiation complex has been developed. This approach could be extended to enable structural modeling and prediction of other bio-molecular complexes. Just as models of individual proteins provide insight into molecular recognition, catalytic mechanism, and substrate specificity, models of protein complexes will provide understanding into the combinatorial rules of cellular regulation and signaling.
Background:
Plasmodium falciparum is the protozoan parasite primarily responsible for more than one million malarial deaths, annually, and is developing resistance to current therapies. Throughout its lifespan, the parasite is subjected to oxidative attack, so Plasmodium antioxidant defences are essential for its survival and are targets for disease control.
Results:
To further understand the molecular aspects of the Plasmodium redox system, we solved 4 structures of Plasmodium peroxiredoxins (Prx). Our study has confirmed PvTrx-Px1 to be a hydrogen peroxide (H2O2)-sensitive peroxiredoxin. We have identified and characterized the novel toroid octameric oligomer of PyTrx-Px1, which may be attributed to the interplay of several factors including: (1) the orientation of the conserved surface/buried arginine of the NNLA(I/L)GRS-loop; and (2) the C-terminal tail positioning (also associated with the aforementioned conserved loop) which facilitates the intermolecular hydrogen bond between dimers (in an A-C fashion). In addition, a notable feature of the disulfide bonds in some of the Prx crystal structures is discussed. Finally, insight into the latter stages of the peroxiredoxin reaction coordinate is gained. Our structure of PyPrx6 is not only in the sulfinic acid (RSO2H) form, but it is also with glycerol bound in a way (not previously observed) indicative of product binding.
Conclusions:
The structural characterization of Plasmodium peroxiredoxins provided herein provides insight into their oligomerization and product binding which may facilitate the targeting of these antioxidant defences. Although the structural basis for the octameric oligomerization is further understood, the results yield more questions about the biological implications of the peroxiredoxin oligomerization, as multiple toroid configurations are now known. The crystal structure depicting the product bound active site gives insight into the overoxidation of the active site and allows further characterization of the leaving group chemistry.
Background:
Protein-DNA interactions play a crucial role in the life of biological organisms in controlling transcription, regulation, as well as DNA recombination and repair. The deep understanding of these processes, which requires the atomic description of the interactions occurring between the proteins and their DNA partners is often limited by the absence of a 3D structure of such complexes.
Results:
In this study, using a method combining sequence homology, structural analogy modeling and biochemical data, we first build the 3D structure of the complex between the poorly-characterized PerR-like regulator Slr1738 and its target DNA, which controls the defences against metal and oxidative stresses in Synechocystis. In a second step, we propose an expanded version of the Slr1738-DNA structure, which accommodates the DNA binding of Slr1738 multimers, a feature likely operating in the complex Slr1738-mediated regulation of stress responses. Finally, in agreement with experimental data we present a 3D-structure of the Slr1738-DNA complex resulting from the binding of multimers of the FUR-like regulator onto its target DNA that possesses internal repeats.
Conclusion:
Using a combination of different types of data, we build and validate a relevant model of the tridimensional structure of a biologically important protein-DNA complex. Then, based on published observations, we propose more elaborated multimeric models that may be biologically important to understand molecular mechanisms.
Background:
Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy) or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy). Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs.
Results:
The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments.
Conclusions:
Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules.
Posted on 22 December 2011 | 1:00 am
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