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1.  Discriminating the native structure from decoys using scoring functions based on the residue packing in globular proteins 
Background
Setting the rules for the identification of a stable conformation of a protein is of utmost importance for the efficient generation of structures in computer simulation. For structure prediction, a considerable number of possible models are generated from which the best model has to be selected.
Results
Two scoring functions, Rs and Rp, based on the consideration of packing of residues, which indicate if the conformation of an amino acid sequence is native-like, are presented. These are defined using the solvent accessible surface area (ASA) and the partner number (PN) (other residues that are within 4.5 Å) of a particular residue. The two functions evaluate the deviation from the average packing properties (ASA or PN) of all residues in a polypeptide chain corresponding to a model of its three-dimensional structure. While simple in concept and computationally less intensive, both the functions are at least as efficient as any other energy functions in discriminating the native structure from decoys in a large number of standard decoy sets, as well as on models submitted for the targets of CASP7. Rs appears to be slightly more effective than Rp, as determined by the number of times the native structure possesses the minimum value for the function and its separation from the average value for the decoys.
Conclusion
Two parameters, Rs and Rp, are discussed that can very efficiently recognize the native fold for a sequence from an ensemble of decoy structures. Unlike many other algorithms that rely on the use of composite scoring function, these are based on a single parameter, viz., the accessible surface area (or the number of residues in contact), but still able to capture the essential attribute of the native fold.
doi:10.1186/1472-6807-9-76
PMCID: PMC2809062  PMID: 20038291
2.  Solution structure of the Equine Infectious Anemia Virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein 
Background
The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.
Results
In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.
Conclusions
These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.
doi:10.1186/1472-6807-9-74
PMCID: PMC2803184  PMID: 20015412
3.  The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from Streptococcus pyogenes 
Background
Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.
Results
We have solved the crystal structure of the gene-product of locus Spy_2152 from S. pyogenes, (PDB:2fu2), and found it to comprise an anti-parallel four-helix bundle that is structurally similar to other bacteriocin immunity proteins. Sequence analyses indicate this protein to be a possible immunity protein protective against class IIa or IIb bacteriocins. However, given that S. pyogenes appears to lack any IIa pediocin-like proteins but does possess class IIb bacteriocins, we suggest this protein confers immunity to IIb-like peptides.
Conclusions
Combined structural, genomic and proteomic analyses have allowed the identification and in silico characterization of a new putative immunity protein from S. pyogenes, possibly the first structure of an immunity protein protective against potential class IIb two-peptide bacteriocins. We have named the two pairs of putative bacteriocins found in S. pyogenes pyogenecin 1, 2, 3 and 4.
doi:10.1186/1472-6807-9-75
PMCID: PMC2806384  PMID: 20017931
4.  TIM-Finder: A new method for identifying TIM-barrel proteins 
Background
The triosephosphate isomerase (TIM)-barrel fold occurs frequently in the proteomes of different organisms, and the known TIM-barrel proteins have been found to play diverse functional roles. To accelerate the exploration of the sequence-structure protein landscape in the TIM-barrel fold, a computational tool that allows sensitive detection of TIM-barrel proteins is required.
Results
To develop a new TIM-barrel protein identification method in this work, we consider three descriptors: a sequence-alignment-based descriptor using PSI-BLAST e-values and bit scores, a descriptor based on secondary structure element alignment (SSEA), and a descriptor based on the occurrence of PROSITE functional motifs. With the assistance of Support Vector Machine (SVM), the three descriptors were combined to obtain a new method with improved performance, which we call TIM-Finder. When tested on the whole proteome of Bacillus subtilis, TIM-Finder is able to detect 194 TIM-barrel proteins at a 99% confidence level, outperforming the PSI-BLAST search as well as one existing fold recognition method.
Conclusions
TIM-Finder can serve as a competitive tool for proteome-wide TIM-barrel protein identification. The TIM-Finder web server is freely accessible at http://202.112.170.199/TIM-Finder/.
doi:10.1186/1472-6807-9-73
PMCID: PMC2803183  PMID: 20003393
5.  Prediction of calcium-binding sites by combining loop-modeling with machine learning 
Background
Protein ligand-binding sites in the apo state exhibit structural flexibility. This flexibility often frustrates methods for structure-based recognition of these sites because it leads to the absence of electron density for these critical regions, particularly when they are in surface loops. Methods for recognizing functional sites in these missing loops would be useful for recovering additional functional information.
Results
We report a hybrid approach for recognizing calcium-binding sites in disordered regions. Our approach combines loop modeling with a machine learning method (FEATURE) for structure-based site recognition. For validation, we compared the performance of our method on known calcium-binding sites for which there are both holo and apo structures. When loops in the apo structures are rebuilt using modeling methods, FEATURE identifies 14 out of 20 crystallographically proven calcium-binding sites. It only recognizes 7 out of 20 calcium-binding sites in the initial apo crystal structures.
We applied our method to unstructured loops in proteins from SCOP families known to bind calcium in order to discover potential cryptic calcium binding sites. We built 2745 missing loops and evaluated them for potential calcium binding. We made 102 predictions of calcium-binding sites. Ten predictions are consistent with independent experimental verifications. We found indirect experimental evidence for 14 other predictions. The remaining 78 predictions are novel predictions, some with intriguing potential biological significance. In particular, we see an enrichment of beta-sheet folds with predicted calcium binding sites in the connecting loops on the surface that may be important for calcium-mediated function switches.
Conclusion
Protein crystal structures are a potentially rich source of functional information. When loops are missing in these structures, we may be losing important information about binding sites and active sites. We have shown that limited loop modeling (e.g. loops less than 17 residues) combined with pattern matching algorithms can recover functions and propose putative conformations associated with these functions.
doi:10.1186/1472-6807-9-72
PMCID: PMC2808310  PMID: 20003365
6.  Splitting statistical potentials into meaningful scoring functions: Testing the prediction of near-native structures from decoy conformations 
Background
Recent advances on high-throughput technologies have produced a vast amount of protein sequences, while the number of high-resolution structures has seen a limited increase. This has impelled the production of many strategies to built protein structures from its sequence, generating a considerable amount of alternative models. The selection of the closest model to the native conformation has thus become crucial for structure prediction. Several methods have been developed to score protein models by energies, knowledge-based potentials and combination of both.
Results
Here, we present and demonstrate a theory to split the knowledge-based potentials in scoring terms biologically meaningful and to combine them in new scores to predict near-native structures. Our strategy allows circumventing the problem of defining the reference state. In this approach we give the proof for a simple and linear application that can be further improved by optimizing the combination of Zscores. Using the simplest composite score () we obtained predictions similar to state-of-the-art methods. Besides, our approach has the advantage of identifying the most relevant terms involved in the stability of the protein structure. Finally, we also use the composite Zscores to assess the conformation of models and to detect local errors.
Conclusion
We have introduced a method to split knowledge-based potentials and to solve the problem of defining a reference state. The new scores have detected near-native structures as accurately as state-of-art methods and have been successful to identify wrongly modeled regions of many near-native conformations.
doi:10.1186/1472-6807-9-71
PMCID: PMC2783033  PMID: 19917096
7.  The structural and functional determinants of the Axin and Dishevelled DIX domains 
Background
The dishevelled and axin genes encode multi-domain proteins that play key roles in WNT signalling. Dishevelled prevents β-catenin degradation by interfering with the interaction of β-catenin with the degradation-mediating Axin-APC-GSK3β complex. This interference leads to an accumulation of cytoplasmic β-catenin, which enters the nucleus and interacts with transcription factors that induce expression of Wnt-target genes. Axin, as a component of the degradation-mediating complex, is a potent negative regulator of Wnt signalling, whereas Dishevelled is a potent activator. Both Dishevelled and Axin possess a DIX (Dishevelled/Axin) domain, which mediates protein-protein interactions, specifically homodimerization.
Results
An evolutionary trace analysis of DIX domains identified conserved residues which, when mapped onto the crystal structure of the Axin DIX domain and a comparative model of the Dishevelled DIX domain, allow their categorization as residues of either structural or functional importance. We identify residues that are structural and functional determinants of the DIX domain fold, as well as those that are specific to homodimerization of Axin and Dishevelled.
Conclusion
This report provides the first explanation of the mutant phenotypes caused by non-synonymous substitutions in the Dishevelled and Axin DIX domain by correlating their presumed functional significance with molecular structure.
doi:10.1186/1472-6807-9-70
PMCID: PMC2780430  PMID: 19909509
8.  Interacting with the biomolecular solvent accessible surface via a haptic feedback device 
Background
From the 1950s computer based renderings of molecules have been produced to aid researchers in their understanding of biomolecular structure and function. A major consideration for any molecular graphics software is the ability to visualise the three dimensional structure of the molecule. Traditionally, this was accomplished via stereoscopic pairs of images and later realised with three dimensional display technologies. Using a haptic feedback device in combination with molecular graphics has the potential to enhance three dimensional visualisation. Although haptic feedback devices have been used to feel the interaction forces during molecular docking they have not been used explicitly as an aid to visualisation.
Results
A haptic rendering application for biomolecular visualisation has been developed that allows the user to gain three-dimensional awareness of the shape of a biomolecule. By using a water molecule as the probe, modelled as an oxygen atom having hard-sphere interactions with the biomolecule, the process of exploration has the further benefit of being able to determine regions on the molecular surface that are accessible to the solvent. This gives insight into how awkward it is for a water molecule to gain access to or escape from channels and cavities, indicating possible entropic bottlenecks. In the case of liver alcohol dehydrogenase bound to the inhibitor SAD, it was found that there is a channel just wide enough for a single water molecule to pass through. Placing the probe coincident with crystallographic water molecules suggests that they are sometimes located within small pockets that provide a sterically stable environment irrespective of hydrogen bonding considerations.
Conclusion
By using the software, named HaptiMol ISAS (available from ), one can explore the accessible surface of biomolecules using a three-dimensional input device to gain insights into the shape and water accessibility of the biomolecular surface that cannot be so easily attained using conventional molecular graphics software.
doi:10.1186/1472-6807-9-69
PMCID: PMC2774689  PMID: 19860901
9.  Partially-supervised protein subclass discovery with simultaneous annotation of functional residues 
Background
The study of functional subfamilies of protein domain families and the identification of the residues which determine substrate specificity is an important question in the analysis of protein domains. One way to address this question is the use of clustering methods for protein sequence data and approaches to predict functional residues based on such clusterings. The locations of putative functional residues in known protein structures provide insights into how different substrate specificities are reflected on the protein structure level.
Results
We have developed an extension of the context-specific independence mixture model clustering framework which allows for the integration of experimental data. As these are usually known only for a few proteins, our algorithm implements a partially-supervised learning approach. We discover domain subfamilies and predict functional residues for four protein domain families: phosphatases, pyridoxal dependent decarboxylases, WW and SH3 domains to demonstrate the usefulness of our approach.
Conclusion
The partially-supervised clustering revealed biologically meaningful subfamilies even for highly heterogeneous domains and the predicted functional residues provide insights into the basis of the different substrate specificities.
doi:10.1186/1472-6807-9-68
PMCID: PMC2777906  PMID: 19857261
10.  Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103 
Background
The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: α-, β-, γ-, δ-, ε-, ζ- CAs, with β-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of β-CA Nce103/YNL036W.
Results
We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 Å resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.
Conclusion
The quaternary structure of Nce103 resembles the typical plant type β-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.
doi:10.1186/1472-6807-9-67
PMCID: PMC2775743  PMID: 19852838
11.  Machine learning integration for predicting the effect of single amino acid substitutions on protein stability 
Background
Computational prediction of protein stability change due to single-site amino acid substitutions is of interest in protein design and analysis. We consider the following four ways to improve the performance of the currently available predictors: (1) We include additional sequence- and structure-based features, namely, the amino acid substitution likelihoods, the equilibrium fluctuations of the alpha- and beta-carbon atoms, and the packing density. (2) By implementing different machine learning integration approaches, we combine information from different features or representations. (3) We compare classification vs. regression methods to predict the sign vs. the output of stability change. (4) We allow a reject option for doubtful cases where the risk of misclassification is high.
Results
We investigate three different approaches: early, intermediate and late integration, which respectively combine features, kernels over feature subsets, and decisions. We perform simulations on two data sets: (1) S1615 is used in previous studies, (2) S2783 is the updated version (as of July 2, 2009) extracted also from ProTherm. For S1615 data set, our highest accuracy using both sequence and structure information is 0.842 on cross-validation and 0.904 on testing using early integration. Newly added features, namely, local compositional packing and the mobility extent of the mutated residues, improve accuracy significantly with intermediate integration. For S2783 data set, we also train regression methods to estimate not only the sign but also the amount of stability change and apply risk-based classification to reject when the learner has low confidence and the loss of misclassification is high. The highest accuracy is 0.835 on cross-validation and 0.832 on testing using only sequence information. The percentage of false positives can be decreased to less than 0.005 by rejecting 10 per cent using late integration.
Conclusion
We find that in both early and late integration, combining inputs or decisions is useful in increasing accuracy. Intermediate integration allows assessing the contributions of individual features by looking at the assigned weights. Overall accuracy of regression is not better than that of classification but it has less false positives, especially when combined with the reject option. The server for stability prediction for three integration approaches and the data sets are available at .
doi:10.1186/1472-6807-9-66
PMCID: PMC2777163  PMID: 19840377
12.  Conformational changes and loose packing promote E. coli Tryptophanase cold lability 
Background
Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.
Results
We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers already at 25°C and even further upon cooling to 2°C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9Å resolution. These apo mutants were found in an open conformation compared to the closed conformation found for P. vulgaris in its holo form. This conformational change is further supported by a high pressure study.
Conclusion
We suggest that cold lability of E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of E. coli Trpase to dimers and P. vulgaris Trpase to monomers.
doi:10.1186/1472-6807-9-65
PMCID: PMC2770544  PMID: 19814824
13.  Molecular models for intrastrand DNA G-quadruplexes 
Background
Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G3Xn1G3Xn2G3Xn3G3 motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.
Results
We have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion.
Conclusion
Molecular models have been generated for a large number of G3Xn1G3Xn2G3Xn3G3 sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.
doi:10.1186/1472-6807-9-64
PMCID: PMC2768733  PMID: 19811654
14.  Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis 
Background
Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog.
Results
Xenavidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin.
Conclusion
The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.
doi:10.1186/1472-6807-9-63
PMCID: PMC2761383  PMID: 19788720
15.  Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor 
Background
The novel A/H1N1 influenza virus, which recently emerged in North America is most closely related to North American H1N1/N2 swine viruses. Until the beginning of 2009, North American swine H1N1/N2 viruses have only sporadically infected humans as dead-end hosts. In 2009 the A/H1N1 virus acquired the capacity to spread efficiently by human to human transmission. The novel A/H1N1 influenza virus has struck thousands of people in more than 70 countries and killed more than 140, representing a public health emergency of international concern. Here we have studied properties of hemagglutinin of A/H1N1 which may modulate virus/receptor interaction.
Results
Analyses by ISM bioinformatics platform of the HA1 protein of North American swine H1N1/N2 viruses and the new A/H1N1 showed that both groups of viruses differed in conserved characteristics that reflect a distinct propensity of these viruses to undergo a specific interaction with swine or human host proteins or receptors. Swine H1N1/N2 viruses that sporadically infected humans featured both the swine and the human interaction pattern. Substitutions F71S, T128S, E302K, M314L in HA1 of swine H1N1 viruses from North America are identified as critical for the human interaction pattern of A/H1N1 and residues D94, D196 and D274 are predicted to be "hot-spots" for polymorphisms which could increase infectivity of A/H1N1 virus. At least one of these residues has already emerged in the A/H1N1 isolates from Spain, Italy and USA. The domain 286-326 was identified to be involved in virus/receptor interaction.
Conclusion
Our results (i) contribute to better understanding of the origin of the novel A/H1N1 influenza virus, (ii) provide a tool for monitoring its molecular evolution (iii) predicts hotspots associated with enhanced infectivity in humans and (iv) identify therapeutic and diagnostic targets for prevention and treatment of A/H1N1 infection.
doi:10.1186/1472-6807-9-62
PMCID: PMC2760557  PMID: 19785758
16.  Mirrors in the PDB: left-handed α-turns guide design with D-amino acids 
Background
Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids.
Results
Propensities for amino acids to occur in contiguous αL helices correlate with published thermodynamic scales for incorporation of D-amino acids into αR helices. Two backbone rules for terminating a left-handed helix are found: an αR conformation is disfavored at the amino terminus, and a βR conformation is disfavored at the carboxy terminus. Helix capping sidechain-backbone interactions are found which are unique to αL helices including an elevated propensity for L-Asn, and L-Thr at the amino terminus and L-Gln, L-Thr and L-Ser at the carboxy terminus.
Conclusion
By examining left-handed α-turns containing L-amino acids, new interaction motifs for incorporating D-amino acids into right-handed α-helices are identified. These will provide a basis for de novo design of novel heterochiral protein folds.
doi:10.1186/1472-6807-9-61
PMCID: PMC2759939  PMID: 19772623
17.  Exploring protein structural dissimilarity to facilitate structure classification 
Background
Classification of newly resolved protein structures is important in understanding their architectural, evolutionary and functional relatedness to known protein structures. Among various efforts to improve the database of Structural Classification of Proteins (SCOP), automation has received particular attention. Herein, we predict the deepest SCOP structural level that an unclassified protein shares with classified proteins with an equal number of secondary structure elements (SSEs).
Results
We compute a coefficient of dissimilarity (Ω) between proteins, based on structural and sequence-based descriptors characterising the respective constituent SSEs. For a set of 1,661 pairs of proteins with sequence identity up to 35%, the performance of Ω in predicting shared Class, Fold and Super-family levels is comparable to that of DaliLite Z score and shows a greater than four-fold increase in the true positive rate (TPR) for proteins sharing the Family level. On a larger set of 600 domains representing 200 families, the performance of Z score improves in predicting a shared Family, but still only achieves about half of the TPR of Ω. The TPR for structures sharing a Super-family is lower than in the first dataset, but Ω performs slightly better than Z score. Overall, the sensitivity of Ω in predicting common Fold level is higher than that of the DaliLite Z score.
Conclusion
Classification to a deeper level in the hierarchy is specific and difficult. So the efficiency of Ω may be attractive to the curators and the end-users of SCOP. We suggest Ω may be a better measure for structure classification than the DaliLite Z score, with the caveat that currently we are restricted to comparing structures with equal number of SSEs.
doi:10.1186/1472-6807-9-60
PMCID: PMC2754988  PMID: 19765314
18.  The Sam domain of the lipid phosphatase Ship2 adopts a common model to interact with Arap3-Sam and EphA2-Sam 
Background
Sterile alpha motif (Sam) domains are small protein modules that can be involved in homotypic or heterotypic associations and exhibit different functions. Previous studies have demonstrated that the Sam domain of the lipid phosphatase Ship2 can hetero-dimerize with the Sam domain of the PI3K effector protein Arap3.
Results
Here, we determine the NMR solution structure of Arap3-Sam and implement a multidisciplinary approach consisting of NMR spectroscopy, ITC (Isothermal Titration Calorimetry), mutagenesis and molecular modeling studies to analyze the interaction between Ship2-Sam and Arap3-Sam. This work reveals that Arap3-Sam may associate with Ship2-Sam by adopting a binding mode common to other Sam domains. This binding mode is identical to what we have very recently observed for the association between Ship2-Sam and the Sam domain from the Ephrin A2 receptor.
Conclusion
Our studies further clarify the structural features that are relevant for Sam-Sam interactions involving Ship2 and give additional hints that could be used for the identification of new molecules able to selectively inhibit Sam-Sam associations.
doi:10.1186/1472-6807-9-59
PMCID: PMC2755476  PMID: 19765305
19.  Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations 
Background
The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K).
Results
Simulation results are consistent with all currently available clinical/experimental evidence. The simulation-derived putative interface, not possibly obtained from static models, between the kinase (JH1) and pseudokinase (JH2) domains of JAK2 provides a platform able to explain the mutational effect for all mutants, including presumably benign control mutants, at the atomic level.
Conclusion
The results and analysis provide structural bases for mutational mechanisms of JAK2, may advance the understanding of JAK2 auto-regulation, and have the potential to lead to therapeutic approaches. Together with recent mutation profiling results demonstrating the breadth of clinically observed JAK2 mutations, our findings suggest that molecular testing/diagnostics of JAK2 should extend beyond V617F and exon 12 mutations, and perhaps should encompass most of the pseudo-kinase domain-coding region.
doi:10.1186/1472-6807-9-58
PMCID: PMC2749040  PMID: 19744331
20.  Low-resolution structural studies of human Stanniocalcin-1 
Background
Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STC1 is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di- or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC50 and "big STC", which molecular weights range from 56 to 135 kDa.
Results
In this study we performed a biochemical and structural analysis of STC1 with the aim of obtaining low resolution structural information about the human STC1, since structural information in this protein family is scarce. We expressed STC1 in both E. coli and insect cells using the baculo virus system with a C-terminal 6 × His fusion tag. From the latter we obtained reasonable amounts of soluble protein. Circular dichroism analysis showed STC1 as a well structured protein with 52% of alpha-helical content. Mass spectroscopy analysis of the recombinant protein allowed to assign the five intramolecular disulfide bridges as well as the dimerization Cys202, thereby confirming the conservation of the disulfide pattern previously described for fish STC1. SAXS data also clearly demonstrated that STC1 adopts a dimeric, slightly elongated structure in solution.
Conclusion
Our data reveal the first low resolution, structural information for human STC1. Theoretical predictions and circular dichroism spectroscopy both suggested that STC1 has a high content of alpha-helices and SAXS experiments revealed that STC1 is a dimer of slightly elongated shape in solution. The dimerization was confirmed by mass spectrometry as was the highly conserved disulfide pattern, which is identical to that found in fish STC1.
doi:10.1186/1472-6807-9-57
PMCID: PMC2744999  PMID: 19712479
21.  Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography 
Background
The lysosomal 66.3 kDa protein from mouse is a soluble, mannose 6-phosphate containing protein of so far unknown function. It is synthesized as a glycosylated 75 kDa precursor that undergoes limited proteolysis leading to a 28 kDa N- and a 40 kDa C-terminal fragment.
Results
In order to gain insight into the function and the post-translational maturation process of the glycosylated 66.3 kDa protein, three crystal structures were determined that represent different maturation states. These structures demonstrate that the 28 kDa and 40 kDa fragment which have been derived by a proteolytic cleavage remain associated. Mass spectrometric analysis confirmed the subsequent trimming of the C-terminus of the 28 kDa fragment making a large pocket accessible, at the bottom of which the putative active site is located. The crystal structures reveal a significant similarity of the 66.3 kDa protein to several bacterial hydrolases. The core αββα sandwich fold and a cysteine residue at the N-terminus of the 40 kDa fragment (C249) classify the 66.3 kDa protein as a member of the structurally defined N-terminal nucleophile (Ntn) hydrolase superfamily.
Conclusion
Due to the close resemblance of the 66.3 kDa protein to members of the Ntn hydrolase superfamily a hydrolytic activity on substrates containing a non-peptide amide bond seems reasonable. The structural homology which comprises both the overall fold and essential active site residues also implies an autocatalytic maturation process of the lysosomal 66.3 kDa protein. Upon the proteolytic cleavage between S248 and C249, a deep pocket becomes solvent accessible, which harbors the putative active site of the 66.3 kDa protein.
doi:10.1186/1472-6807-9-56
PMCID: PMC2739207  PMID: 19706171
22.  The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation 
Background
The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å.
Results
The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects.
Conclusion
The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions.
doi:10.1186/1472-6807-9-55
PMCID: PMC2737543  PMID: 19698123
23.  The cyanobacterial cell division factor Ftn6 contains an N-terminal DnaD-like domain 
Background
DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation.
Results
In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain), exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271). We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions.
Conclusion
Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.
doi:10.1186/1472-6807-9-54
PMCID: PMC2736966  PMID: 19698108
24.  Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames) 
Background
Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (AlrBax), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.
Results
B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.
Conclusion
The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.
doi:10.1186/1472-6807-9-53
PMCID: PMC2743695  PMID: 19695097
25.  A tool for calculating binding-site residues on proteins from PDB structures 
Background
In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB) that consists of the protein of interest and its interacting partner(s) and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice.
Results
In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP . For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues.
Conclusion
The developed tool is very useful for the research on protein binding site analysis and prediction.
doi:10.1186/1472-6807-9-52
PMCID: PMC2728722  PMID: 19650927

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