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The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine 5′-monophosphate (AMP). The resulting AMPylation prevented the interaction of Rho GTPases with downstream effectors, thereby inhibiting actin assembly in the infected cell. Eukaryotic proteins were also directly modified with AMP, potentially expanding the repertoire of posttranslational modifications for molecular signaling.

Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [(3)H]cholesterol (K(d), 130 nM) and [(3)H]25-hydroxycholesterol (25-HC, K(d), 10 nM) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [(3)H]cholesterol, but weakly for [(3)H]25-HC binding. Binding of [(3)H]cholesterol but not [(3)H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [(3)H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [(3)H]cholesterol and [(3)H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.

X-ray crystallographic protein structures often contain disordered regions that are observed as missing electron density. Diffraction data may give little or no direct evidence as to the specific nature of disordered regions. We have developed a weighted window-based disorder predictor optimized using crystallographic data. Performance of a predictor is strongly influenced by chain termini. Optimized score adjustment values for amino- and carboxy-terminal positions demonstrate a simple, monotonic relationship between disorder and residue distance from termini. This optimized disorder predictor performs similarly to DISOPRED2 on crystallographically disordered regions. Data-optimized residue disorder propensities show strong linear correlation with experimentally determined amino acid transfer energies between water and hydrogen-bonding organic solvents, which primarily reflect residue hydrophobicity (exemplified by the Nozaki-Tanford hydrophobicity scale). Disorder propensities do not correlate as well with transfer energies between water and apolar solvents, which primarily reflect a different hydropathic property: residue hydrophilicity (also reflected by the Kyte-Doolittle hydropathy scale). Our results suggest that while hydrophobic side-chain interactions are primarily involved in determining stability of the folded conformation, hydrogen bonding, and similar polar interactions are primarily involved in conformational and interaction specificity.

We present an assessment of 'template-free modeling' (FM) in CASP11and ROLL. Community-wide server performance suggested the use of automated scores similar to previous CASPs would provide a good system of evaluating performance, even in the absence of comprehensive manual assessment. The CASP11 FM category included several outstanding examples, including successful prediction by the Baker group of a 256-residue target (T0806-D1) that lacked sequence similarity to any existing template. The top server model prediction by Zhang's Quark, which was apparently selected and refined by several manual groups, encompassed the entire fold of target T0837-D1. Methods from the same two groups tended to dominate overall CASP11 FM and ROLL rankings. Comparison of top FM predictions with those from the previous CASP experiment revealed progress in the category, particularly reflected in high prediction accuracy for larger protein domains. FM prediction models for two cases were sufficient to provide functional insights that were otherwise not obtainable by traditional sequence analysis methods. Importantly, CASP11 abstracts revealed that alignment-based contact prediction methods brought about much of the CASP11 progress, producing both of the functionally relevant models as well as several of the other outstanding structure predictions. These methodological advances enabled de novo modeling of much larger domain structures than was previously possible and allowed prediction of functional sites. Proteins 2016; 84(Suppl 1):51-66. © 2015 Wiley Periodicals, Inc.

The prediction of the structures of proteins without detectable sequence similarity to any protein of known structure remains an outstanding scientific challenge. Here we report significant progress in this area. We first describe de novo blind structure predictions of unprecendented accuracy we made for two proteins in large families in the recent CASP11 blind test of protein structure prediction methods by incorporating residue–residue co-evolution information in the Rosetta structure prediction program. We then describe the use of this method to generate structure models for 58 of the 121 large protein families in prokaryotes for which three-dimensional structures are not available. These models, which are posted online for public access, provide structural information for the over 400,000 proteins belonging to the 58 families and suggest hypotheses about mechanism for the subset for which the function is known, and hypotheses about function for the remainder. DOI: http://dx.doi.org/10.7554/eLife.09248.001, eLife digest Proteins are long chains made up of small building blocks called amino acids. These chains fold up in various ways to form the three-dimensional structures that proteins need to be able work properly. Therefore, to understand how a protein works it is important to determine its structure, but this is very challenging. It is possible to predict the structure of a protein with high accuracy if previous experiments have revealed the structure of a similar protein. However, for almost half of all known families of proteins, there are currently no members whose structures have been solved. The three-dimensional shape of a protein is determined by interactions between various amino acids. During evolution, the structure and activity of proteins often remain the same across species, even if the amino acid sequences have changed. This is because pairs of amino acids that interact with each other tend to ‘co-evolve’; that is, if one amino acid changes, then the second amino acid also changes in order to accommodate it. By identifying these pairs of co-evolving amino acids, it is possible to work out which amino acids are close to each other in the three-dimensional structure of the protein. This information can be used to generate a structural model of a protein using computational methods. Now, Ovchinnikov et al. developed a new method to predict the structures of proteins that combines data on the co-evolution of amino acids, as identified by GREMLIN with the structural prediction software called Rosetta. A community-wide experiment called CASP—which tests different methods of protein prediction—showed that, in two cases, this new method works much better than anything previously used to predict the structures of proteins. Ovchinnikov et al. then used this method to make models for proteins belonging to 58 different protein families with currently unknown structures. These predictions were found to be highly accurate and the protein families each have thousands of members, so Ovchinnikov et al.'s findings are expected to be useful to researchers in a wide variety of research areas. A future challenge is to extend these methods to the many protein families that have hundreds rather than thousands of members. DOI: http://dx.doi.org/10.7554/eLife.09248.002

The type VI secretion system (T6SS) is a widespread protein secretion apparatus used by Gram-negative bacteria to deliver toxic effector proteins into adjacent bacterial or host cells. Here, we uncovered a role in interbacterial competition for the two T6SSs encoded by the marine pathogen Vibrio alginolyticus. Using comparative proteomics and genetics, we identified their effector repertoires. In addition to the previously described effector V12G01_02265, we identified three new effectors secreted by T6SS1, indicating that the T6SS1 secretes at least four antibacterial effectors, of which three are members of the MIX-effector class. We also showed that the T6SS2 secretes at least three antibacterial effectors. Our findings revealed that many MIX-effectors belonging to clan V are “orphan” effectors that neighbor mobile elements and are shared between marine bacteria via horizontal gene transfer. We demonstrated that a MIX V-effector from V. alginolyticus is a functional T6SS effector when ectopically expressed in another Vibrio species. We propose that mobile MIX V-effectors serve as an environmental reservoir of T6SS effectors that are shared and used to diversify antibacterial toxin repertoires in marine bacteria, resulting in enhanced competitive fitness., Author Summary The bacterial type VI secretion system (T6SS) is a contact-dependent protein secretion apparatus that is emerging as a major component of interbacterial competition in the environment. The bacterium Vibrio alginolyticus is a pathogen of marine animals and a causal agent of wound infections, otitis, and gastroenteritis in humans. In this study, we provide a comprehensive characterization of the environmental regulation, antibacterial activities, and secreted effector repertoires of the two T6SSs found in this pathogen. We also identify a subset of T6SS effectors that appear to be mobile and shared between marine bacteria that can interact with each other in aquatic environments. Our findings suggest that bacteria can incorporate T6SS effectors from competitors in the environment. These newly acquired toxins may be used to expand and diversify T6SS effector repertoires and enhance bacterial fitness.

Inference of homology from protein sequences provides an essential tool for analyzing protein structure, function, and evolution. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. In computer science a search engine can be improved by considering networks of known relationships within the search database. Here, we apply this idea to protein-sequence–based homology search and show that it dramatically enhances the search accuracy. Our new method, COMPADRE (COmparison of Multiple Protein sequence Alignments using Database RElationships) assesses the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit’s known homologs. This approach increases detection quality, boosting the precision rate from 18% to 83% at half-coverage of all database homologs. The increased precision rate allows detection of a large fraction of protein structural relationships, thus providing structure and function predictions for previously uncharacterized proteins. Our results suggest that this general approach is applicable to a wide variety of methods for detection of biological similarities. The web server is available at prodata.swmed.edu/compadre.

Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until only recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases which have N-terminal signal sequence and localize to the lumen of the endoplasmic reticulum and the Golgi apparatus. One of these kinases known as Fam20C is the Golgi casein kinase and it phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and many other secretory proteins including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) implicated in biomineralization. Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Another family member, Fam20B, is a xylose kinase important in the maturation of proteoglycans. The function of other FAM 20C family members will also be addressed.

Chameleon sequences (ChSeqs) refer to sequence strings of identical amino acids that can adopt different conformations in protein structures. Researchers have detected and studied ChSeqs to understand the interplay between local and global interactions in protein structure formation. The different secondary structures adopted by one ChSeq challenge sequence-based secondary structure predictors. With increasing numbers of available Protein Data Bank structures, we here identify a large set of ChSeqs ranging from 6 to 10 residues in length. The homologous ChSeqs discovered highlight the structural plasticity involved in biological function. When compared with previous studies, the set of unrelated ChSeqs found represents an about 20-fold increase in the number of detected sequences, as well as an increase in the longest ChSeq length from 8 to 10 residues. We applied secondary structure predictors on our ChSeqs and found that methods based on a sequence profile outperformed methods based on a single sequence. For the unrelated ChSeqs, the evolutionary information provided by the sequence profile typically allows successful prediction of the prevailing secondary structure adopted in each protein family. Our dataset will facilitate future studies of ChSeqs, as well as interpretations of the interplay between local and nonlocal interactions. A user-friendly web interface for this ChSeq database is available at prodata.swmed.edu/chseq.

Restriction endonucleases and other nucleic acid cleaving enzymes form a large and extremely diverse superfamily that display little sequence similarity despite retaining a common core fold responsible for cleavage. The lack of significant sequence similarity between protein families makes homology inference a challenging task and hinders new family identification with traditional sequence-based approaches. Using the consensus fold recognition method Meta-BASIC that combines sequence profiles with predicted protein secondary structure, we identify nine new restriction endonuclease-like fold families among previously uncharacterized proteins and predict these proteins to cleave nucleic acid substrates. Application of transitive searches combined with gene neighborhood analysis allow us to confidently link these unknown families to a number of known restriction endonuclease-like structures and thus assign folds to the uncharacterized proteins. Finally, our method identifies a novel restriction endonuclease-like domain in the C-terminus of RecC that is not detected with structure-based searches of the existing PDB database.

Using top-of-the-range fold-recognition methods, we have assigned a thioredoxin-like structure to a family of previously uncharacterized hypothetical proteins of bacterial origin. The DCC family, named after the conserved N-terminal DxxCxxC motif, encompasses proteins of unknown function from DUF393 (in Pfam database) and COG3011. The presence of two invariant potentially catalytic cysteine residues indicates that DCC proteins function as thiol-disulfide oxidoreductases.

We present an overview of the fifth round of Critical Assessment of Protein Structure Prediction (CASP5) fold recognition category. Prediction models were evaluated by using six different structural measures and four different alignment measures, and these scores were compared to those assigned manually over a diverse subset of target domains. Scores were combined to compare overall performance of participating groups and to estimate rank significance. The methods used by a few groups outperformed all other methods in terms of the evaluated criteria and could be considered state-of-the-art in structure prediction. We discuss a few examples of difficult fold recognition targets to highlight the progress of ab initio-type methods on difficult structure analogs and the difficulties of predicting multidomain targets and selecting prediction models. We also compared the results of manual groups to those of automatic servers evaluated in parallel by CAFASP, showing that the top performing automated server structure predictions approached those of the best manual predictors.

The carboxyl-terminal domain of phospholipase C-beta is required for its stimulation by Galpha(q) and for its Galpha(q)-specific GTPase-activating protein (GAP) activity. We subjected this domain to a combination of deletion and alanine/glycine scanning mutagenesis to detect mutations that would inhibit either responsiveness to G(q) or G(q) GAP activity. Most mutations that altered either response or GAP activity diminished both in parallel. Many of these mutations map at the interface at which the carboxyl-terminal domain was recently shown to form a dimer (Singer, A. U., et al. (2001) Nat. Struct. Biol., 9, 32-36). Most others clustered in an area that is a plausible Galpha(q) binding site. In addition, one mutation that differentially inhibited GAP activity relative to responsiveness to Galpha(q) mapped in this region at a location modeled to be in close contact with the switch II region of Galpha(q). This is the site at which RGS proteins are thought to exert their GAP activity. Last, a deletion mutation differentially inhibited the response of phospholipase C-beta1 to Galpha(q) without blocking GAP activity. Its location in the molecule suggests that moving the attachment point of the catalytic domain can disrupt its ability to be activated by Galpha(q).

Fumarate hydratase (FH) is a tumor suppressor, but how it acts is unclear. Two reports in this issue of Cancer Cell reveal that FH-deficiency leads to succination of Keap1, stabilization of Nrf2, and induction of stress-response genes including HMOX1, which is important for the survival of FH-deficient cells.

Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies., Author Summary Protein structural domain databases offer a vital resource for structural bioinformatics. These databases provide functional inference for homologous structures, supply templates for structural prediction experiments, and differentiate between homologs and analogs. The rate of structure determination and deposition has increased dramatically over recent years, overwhelming the ability of current classifications to incorporate all new structures. We have developed a fast and reliable methodology for updating domain databases automatically, and created a revised hierarchy for domain classification that emphasizes evolutionary relationships. By classifying all known structures in our database with continuing automatic updates, we provide an up-to-date alternative to current resources. We illustrate several concepts that guided our classification scheme with examples of homology between domains in ECOD that are not observed in other resources.

To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.

Trypanosoma cruzi S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the pyruvoyl-dependent decarboxylation of S-adenosylmethionine (AdoMet), which is an important step in the biosynthesis of polyamines. The time course of the AdoMetDC reaction under single-turnover conditions was measured to determine the rate of the slowest catalytic step up to and including decarboxylation. Analysis of this single-turnover data yields an apparent second-order rate constant for this reaction of 3300 M(-1) s(-1) in the presence of putrescine, which corresponds to a catalytic rate of6 s(-1). This rate is minimally 100-fold faster than the steady-state rate suggesting that product release, which includes Schiff base hydrolysis, limits the overall reaction. AdoMetDC exhibits an inverse solvent isotope effect on the single-turnover kinetics, and the pH profile predicts a pK(a) of 8.9 for the basic limb. These results are consistent with a Cys residue functioning as a general acid in the rate-determining step of the single-turnover reaction. Mutation of Cys-82 to Ala reduces the rate of the single turnover reaction to 11 M(-1) s(-1) in the presence of putrescine. Further, a solvent isotope effect is not observed for the mutant enzyme. Reduction of the wild-type enzyme with cyanoborohydride traps the Schiff base between the enzyme and decarboxylated substrate, while little Schiff base species of either substrate or product was trapped with the C82A mutant. These data suggest that Cys-82 functions as a general acid/base to catalyze Schiff base formation and hydrolysis. The solvent isotope and pH effects are mirrored in single-turnover analysis of reactions without the putrescine activator, yielding an apparent second-order rate constant of 150 M(-1) s(-1). The presence of putrescine increases the single-turnover rate by 20-fold, while it has relatively little effect on the affinity of the enzyme for product. Therefore, putrescine likely activates the T. cruzi AdoMetDC enzyme by accelerating the rate of Schiff base exchange.

The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa (alpha) and 10 kDa (beta). The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction. To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator. In the presence of fully saturating putrescine, k(cat) increases by 9-fold over the unactivated rate to 0.06 s(-1). The model derived Km for AdoMet is 0.05 mM in the absence of putrescine and the model-derived Kd for putrescine binding to free enzyme is 2.5 mM. The Km for AdoMet increases by alpha 2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine activates the T. cruzi enzyme to a similar extent as putrescine.

Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells.

Tuberculosis, caused by Mycobacterium tuberculosis, remains a devastating human infectious disease, causing two million deaths annually. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, Cor) leads to marked CO sensitivity. Heterologous expression of Cor in Escherichia coli rescued it from CO toxicity. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism., IMPORTANCE Macrophages produce a variety of antimicrobial molecules, including nitric oxide (NO), hydrogen peroxide (H2O2), and acid (H+), that serve to kill engulfed bacteria. In addition to these molecules, human and mouse macrophages also produce carbon monoxide (CO) gas by the heme oxygenase (HO1) enzyme. We observed that, in contrast to other bacteria, mycobacteria are resistant to CO, suggesting that this might be an evolutionary adaptation of mycobacteria for survival within macrophages. We screened a panel of ~2,500 M. tuberculosis mutants to determine which genes are required for survival of M. tuberculosis in the presence of CO. Within this panel, we identified one such gene, cor, that specifically confers CO resistance. Importantly, we found that the ability of M. tuberculosis cells carrying a mutated copy of this gene to cause tuberculosis in a mouse disease model is significantly attenuated. This indicates that CO resistance is essential for mycobacterial survival in vivo.

Two errors were made in this article. First, a reference was omitted from the second sentence of the fourth paragraph in the Introduction section. The sentence should read: SidD lacks any obvious sequence homology with the AT-N or other known proteins, although fold recognition analysis of the N-terminal portion of SidD predicted limited resemblance with members of the metal-dependent protein phosphatase (PPM) family (Rigden 2011). The reference is: Rigden DJ. (2011) Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD. FEBS Lett. 585:2749-54. doi: 10.1016/j.febslet.2011.08.006. Second, there were some omissions from the acknowledgements section, which should instead read as follows: We thank K. B. Decker, Y. Lin, and A.H. Gaspar for comments on the manuscript, and A. Vidaurrazaga for assistance with biochemical experiments. This study made use of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) under the Block Allocation Group (BAG) MX1420, the Diamond Light Source (Oxfordshire, UK) under the rapid access mode MX7512 and BAG MX8302, the beamline PROXIMA1 at the SOLEIL synchrotron (Saint-Aubin, France), the X-ray crystallography platform and proteomics platform (member of CIBERehd and ProteoRed-ISCIII) at CIC bioGUNE (Derio, Spain), and the SGIker analytical facility at UPV/EHU (Leioa, Spain). We thank all the staff from these facilities for technical and human support. Access to synchrotron facilities was part funded by the the BioStruct-X project (proposal number 2460).

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors are implicated in development and tumorigenesis and dual inhibitors like sunitinib are prescribed for cancer treatment. While mammalian VEGF and PDGF receptors are present in multiple isoforms and heterodimers, Drosophila encodes one ancestral PDGF/VEGF receptor, PVR. We identified PVR in an unbiased cell-based RNA interference (RNAi) screen of all Drosophila kinases and phosphatases for novel regulators of TORC1. PVR is essential to sustain target of rapamycin complex 1 (TORC1) and extracellular signal-regulated kinase (ERK) activity in cultured insect cells and for maximal stimulation by insulin. CG32406 (henceforth, PVRAP, for PVR adaptor protein), an Src homology 2 (SH2) domain-containing protein, binds PVR and is required for TORC1 activation. TORC1 activation by PVR involves Tsc1/Tsc2 and, in a cell-type-dependent manner, Lobe (ortholog of PRAS40). PVR is required for cell survival in vitro, and both PVR and TORC1 are necessary for hemocyte expansion in vivo. Constitutive PVR activation induces tumor-like structures that exhibit high TORC1 activity. Like its mammalian orthologs, PVR is inhibited by sunitinib, and sunitinib treatment phenocopies PVR loss in hemocytes. Sunitinib inhibits TORC1 in insect cells, and sunitinib-mediated TORC1 inhibition requires an intact Tsc1/Tsc2 complex. Sunitinib similarly inhibited TORC1 in human endothelial cells in a Tsc1/Tsc2-dependent manner. Our findings provide insight into the mechanism of action of PVR and may have implications for understanding sunitinib sensitivity and resistance in tumors. © 2013, American Society for Microbiology.

Background The size of the protein sequence database has been exponentially increasing due to advances in genome sequencing. However, experimentally characterized proteins only constitute a small portion of the database, such that the majority of sequences have been annotated by computational approaches. Current automatic annotation pipelines inevitably introduce errors, making the annotations unreliable. Instead of such error-prone automatic annotations, functional interpretation should rely on annotations of ‘reference proteins’ that have been experimentally characterized or manually curated. Results The Seq2Ref server uses BLAST to detect proteins homologous to a query sequence and identifies the reference proteins among them. Seq2Ref then reports publications with experimental characterizations of the identified reference proteins that might be relevant to the query. Furthermore, a plurality-based rating system is developed to evaluate the homologous relationships and rank the reference proteins by their relevance to the query. Conclusions The reference proteins detected by our server will lend insight into proteins of unknown function and provide extensive information to develop in-depth understanding of uncharacterized proteins. Seq2Ref is available at: http://prodata.swmed.edu/seq2ref.

The Small Cabbage White (Pieris rapae) is originally a Eurasian butterfly. Being accidentally introduced into North America, Australia, and New Zealand a century or more ago, it spread throughout the continents and rapidly established as one of the most abundant butterfly species. Although it is a serious pest of cabbage and other mustard family plants with its caterpillars reducing crops to stems, it is also a source of pierisin, a protein unique to the Whites that shows cytotoxicity to cancer cells. To better understand the unusual biology of this omnipresent agriculturally and medically important butterfly, we sequenced and annotated the complete genome from USA specimens. At 246 Mbp, it is among the smallest Lepidoptera genomes reported to date. While 1.5% positions in the genome are heterozygous, they are distributed highly non-randomly along the scaffolds, and nearly 20% of longer than 1000 base-pair segments are SNP-free (median length: 38000 bp). Computational simulations of population evolutionary history suggest that American populations started from a very small number of introduced individuals, possibly a single fertilized female, which is in agreement with historical literature. Comparison to other Lepidoptera genomes reveals several unique families of proteins that may contribute to the unusual resilience ofPieris. The nitrile-specifier proteins divert the plant defense chemicals to non-toxic products. The apoptosis-inducing pierisins could offer a defense mechanism against parasitic wasps. While only two pierisins fromPieris rapaewere characterized before, the genome sequence revealed eight, offering additional candidates as anti-cancer drugs. The reference genome we obtained lays the foundation for future studies of the Cabbage White and other Pieridae species.

Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Km for ornithine by 2000-fold, with little effect on kcat, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases kcat 50-fold; however, substitution of N-methylpyridoxal-5'-phosphate for pyridoxal-5'-phosphate as the cofactor in the reaction restores the kcat of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a parasitic Gram-negative bacterium that is closely associated with Huanglongbing (HLB), a worldwide citrus disease. Given the difficulty in culturing the bacterium and thus in its experimental characterization, computational analyses of the whole Ca. L. asiaticus proteome can provide much needed insights into the mechanisms of the disease and guide the development of treatment strategies. In this study, we applied state-of-the-art sequence analysis tools to every Ca. L. asiaticus protein. Our results are available as a public website at http://prodata.swmed.edu/liberibacter_asiaticus/. In particular, we manually curated the results to predict the subcellular localization, spatial structure and function of all Ca. L. asiaticus proteins (http://prodata.swmed.edu/liberibacter_asiaticus/curated/ ). This extensive information should facilitate the study of Ca. L. asiaticus proteome function and its relationship to disease. Pilot studies based on the information from our website have revealed several potential virulence factors, discussed herein.

WNK1 [with no lysine (K)-1] is a 250-kDa serine/threonine protein kinase involved in the maintenance of cellular salt levels and is directly linked to a hereditary form of hypertension. Here, we report the solution NMR structure of the autoinhibitory domain of WNK1 (WNK1-AI), a small regulatory subunit that lies immediately C-terminal of the kinase domain. We show that this domain is a homolog of the RFXV-binding PASK/FRAY homology 2 (PF2) domain found in OSR (oxidative stress responsive) and SPAK (serine/threonine proline-alanine-rich) kinases, which are substrates of WNK1. The WNK1-AI has a circularly permuted topology relative to the OSR1-PF2 domain. Nevertheless, like PF2 domains, WNK1-AI binds peptides that contain an RFXV motif with micromolar affinities as assessed by changes in (1)H,(15)N heteronuclear single quantum coherence spectra. Mutations to the WNK1-AI and binding peptides confirm a similar binding mode.

The Real McCoy Some secreted proteins are phosphorylated, the most prominent example being the milk protein casein, but the enzymes that catalyze such phosphorylation have not been identified. (The proteins known as “casein kinases” are in fact cytosolic proteins and do not mediate physiological phosphorylation of casein.) Tagliabracci et al. (p. 1150 , published online 10 May) searched for a human protein with the characteristics expected of a secretory protein kinase and identified Fam20C. Mutations in the gene encoding Fam20C cause defects in bone formation. Furthermore, the consensus sequence for Fam20C phosphorylation was found in several secreted proteins that function in biomineralization. Thus, Fam20C appears to be the “real” casein kinase and to function in bone physiology.

Motivation: Manual inspection has been applied to and is well accepted for assessing critical assessment of protein structure prediction (CASP) free modeling (FM) category predictions over the years. Such manual assessment requires expertise and significant time investment, yet has the problems of being subjective and unable to differentiate models of similar quality. It is beneficial to incorporate the ideas behind manual inspection to an automatic score system, which could provide objective and reproducible assessment of structure models. Results: Inspired by our experience in CASP9 FM category assessment, we developed an automatic superimposition independent method named Quality Control Score (QCS) for structure prediction assessment. QCS captures both global and local structural features, with emphasis on global topology. We applied this method to all FM targets from CASP9, and overall the results showed the best agreement with Manual Inspection Scores among automatic prediction assessment methods previously applied in CASPs, such as Global Distance Test Total Score (GDT_TS) and Contact Score (CS). As one of the important components to guide our assessment of CASP9 FM category predictions, this method correlates well with other scoring methods and yet is able to reveal good-quality models that are missed by GDT_TS. Availability: The script for QCS calculation is available at http://prodata.swmed.edu/QCS/. Contact: grishin@chop.swmed.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

SummaryRIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here, we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.

The Critical assessment of protein structure prediction round 9 (CASP9) aimed to evaluate predictions for 129 experimentally determined protein structures. To assess tertiary structure predictions, these target structures were divided into domain-based evaluation units that were then classified into two assessment categories: template based modeling (TBM) and template free modeling (FM). CASP9 targets were split into domains of structurally compact evolutionary modules. For the targets with more than one defined domain, the decision to split structures into domains for evaluation was based on server performance. Target domains were categorized based on their evolutionary relatedness to existing templates as well as their difficulty levels indicated by server performance. Those target domains with sequence-related templates and high server prediction performance were classified as TMB, whereas those targets without identifiable templates and low server performance were classified as FM. However, using these generalizations for classification resulted in a blurred boundary between CASP9 assessment categories. Thus, the FM category included those domains without sequence detectable templates (25 target domains) as well as some domains with difficult to detect templates whose predictions were as poor as those without templates (five target domains). Several interesting examples are discussed, including targets with sequence related templates that exhibit unusual structural differences, targets with homologous or analogous structure templates that are not detectable by sequence, and targets with new folds.

ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity.

Polyamine biosynthesis is extensively regulated in cells by multiple mechanisms, including regulation of enzyme activity posttranslationally. The identified regulatory factors include both small molecules and regulatory proteins, and the mechanisms vary in different species across the evolutionary tree. Based on this diversity of mechanism, it is likely that regulatory factors of the pathway remain unidentified in many species. This article focuses on methods for identifying novel regulatory factors of polyamine biosynthesis as illustrated by the discovery of a novel protein activator of the key biosynthetic enzyme S-adenosylmethionine decarboxylase in the protozoan trypanosomatid parasites.

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP.

Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the alpha-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N(1)-aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the alpha-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some alpha-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.

Similarity between protein sequences is usually predictive of similarity in structures. However, in some rare cases protein domains with significant sequence similarity adopt different structures. Here, we carry out a survey of protein domain pairs with high sequence similarity (measured by HHsearch probability) and low structural similarity (measured by Dali Z-score), aiming to identify the reasons for this discordance. Besides methodological problems with either sequences or structures of domains, we find and describe novel examples of homologs with structural changes.

We describe predictions made using the Rosetta structure prediction methodology for the Eighth Critical Assessment of Techniques for Protein Structure Prediction. Aggressive sampling and all-atom refinement were carried out for nearly all targets. A combination of alignment methodologies was used to generate starting models from a range of templates, and the models were then subjected to Rosetta all atom refinement. For the 64 domains with readily identified templates, the best submitted model was better than the best alignment to the best template in the Protein Data Bank for 24 cases, and improved over the best starting model for 43 cases. For 13 targets where only very distant sequence relationships to proteins of known structure were detected, models were generated using the Rosetta de novo structure prediction methodology followed by all-atom refinement; in several cases the submitted models were better than those based on the available templates. Of the 12 refinement challenges, the best submitted model improved on the starting model in seven cases. These improvements over the starting template-based models and refinement tests demonstrate the power of Rosetta structure refinement in improving model accuracy.

Protein structural domains are necessary for understanding evolution and protein folding, and may vary widely from functional and sequence based domains. Although, various structural domain databases exist, defining domains for some proteins is non-trivial, and definitions of their domain boundaries are not available. Here, we present a novel database of manually defined structural domains for a representative set of proteins from the SCOP "multi-domain proteins" class. (http://prodata.swmed.edu/multidom/). We consider our domains as mobile evolutionary units, which may rearrange during protein evolution. Additionally, they may be visualized as structurally compact and possibly independently folding units. We also found that representing domains as evolutionary and folding units do not always lead to a unique domain definition. However, unlike existing databases, we retain and refine these "alternate" domain definitions after careful inspection of structural similarity, functional sites and automated domain definition methods. We provide domain definitions, including actual residue boundaries, for proteins that well known databases like SCOP and CATH do not attempt to split. Our alternate domain definitions are suitable for sequence and structure searches by automated methods. Additionally, the database can be used for training and testing domain delineation algorithms. Since our domains represent structurally compact evolutionary units, the database may be useful for studying domain properties and evolution.

Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [(3)H]cholesterol (K(d), 130 nM) and [(3)H]25-hydroxycholesterol (25-HC, K(d), 10 nM) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [(3)H]cholesterol, but weakly for [(3)H]25-HC binding. Binding of [(3)H]cholesterol but not [(3)H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [(3)H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [(3)H]cholesterol and [(3)H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.

Background The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. Results The structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. Conclusion SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric "lids" that cap the central cavity of the toroid on either side and provide only small openings to allow regulated entry of small molecules into the occluded chamber. The presence of the electron density of the bound ligand may provide important clues on the likely function of NIF3-like proteins.

Eukaryotic protein trafficking pathways require specific transfer of cargo vesicles to different target organelles. A number of vesicle trafficking and membrane fusion components participate in this process, including various tethering factor complexes that interact with small GTPases prior to SNARE-mediated vesicle fusion. In Saccharomyces cerevisiae a protein complex of Mon1 and Ccz1 functions with the small GTPase Ypt7 to mediate vesicle trafficking to the vacuole. Mon1 belongs to DUF254 found in a diverse range of eukaryotic genomes, while Ccz1 includes a CHiPS domain that is also present in a known human protein trafficking disorder gene (HPS-4). The present work identifies the CHiPS domain and a sequence region from another trafficking disorder gene (HPS-1) as homologs of an N-terminal domain from DUF254. This link establishes the evolutionary conservation of a protein complex (HPS-1/HPS-4) that functions similarly to Mon1/Ccz1 in vesicle trafficking to lysosome-related organelles of diverse eukaryotic species. Furthermore, the newly identified DUF254 domain is a distant homolog of the mu-adaptin longin domain found in clathrin adapter protein (AP) complexes of known structure that function to localize cargo protein to specific organelles. In support of this fold assignment, known longin domains such as the AP complex sigma-adaptin, the synaptobrevin N-terminal domains sec22 and Ykt6, and the srx domain of the signal recognition particle receptor also regulate vesicle trafficking pathways by mediating SNARE fusion, recognizing specialized compartments, and interacting with small GTPases that resemble Ypt7.

Using Meta-BASIC, a highly sensitive method for detection of distant similarity between proteins, we have identified another potential PD-(D/E)XK endonuclease in human herpesvirus 1 (HHV-1) encoded by the UL24 gene. The universal presence of UL24 in completed herpesviral genomes of three major subfamilies, Alphaherpesvirinae , Betaherpesvirinae , and Gammaherpesvirinae , suggests a fundamental role for this predicted PD-(D/E)XK endonuclease activity in the viral life cycle.

Site-2 proteases (S2Ps) form a large family of membrane-embedded metalloproteases that participate in cellular signaling pathways through sequential cleavage of membrane-tethered substrates. Using sequence similarity searches, we extend the S2P family to include remote homologs that help define a conserved structural core consisting of three predicted transmembrane helices with traditional metalloprotease functional motifs and a previously unrecognized motif (GxxxN/S/G). S2P relatives were identified in genomes from Bacteria, Archaea, and Eukaryota including protists, plants, fungi, and animals. The diverse S2P homologs divide into several groups that differ in various inserted domains and transmembrane helices. Mammalian S2P proteases belong to the major ubiquitous group and contain a PDZ domain. Sequence and structural analysis of the PDZ domain support its mediating the sequential cleavage of membrane-tethered substrates. Finally, conserved genomic neighborhoods of S2P homologs allow functional predictions for PDZ-containing transmembrane proteases in extra-cytoplasmic stress response and lipid metabolism.

Motivation: A number of methods have been developed to predict functional specificity determinants in protein families based on sequence information. Most of these methods rely on pre-defined functional subgroups. Manual subgroup definition is difficult because of the limited number of experimentally characterized subfamilies with differing specificity, while automatic subgroup partitioning using computational tools is a non-trivial task and does not always yield ideal results. Results: We propose a new approach SPEL (specificity positions by evolutionary likelihood) to detect positions that are likely to be functional specificity determinants. SPEL, which does not require subgroup definition, takes a multiple sequence alignment of a protein family as the only input, and assigns a P-value to every position in the alignment. Positions with low P-values are likely to be important for functional specificity. An evolutionary tree is reconstructed during the calculation, and P-value estimation is based on a random model that involves evolutionary simulations. Evolutionary log-likelihood is chosen as a measure of amino acid distribution at a position. To illustrate the performance of the method, we carried out a detailed analysis of two protein families (LacI/PurR and G protein α subunit), and compared our method with two existing methods (evolutionary trace and mutual information based). All three methods were also compared on a set of protein families with known ligand-bound structures. Availability: SPEL is freely available for non-commercial use. Its pre-compiled versions for several platforms and alignments used in this work are available at Contact: grishin@chop.swmed.edu. Supplementary information: Supplementary materials are available at

Using a recently developed program (SCOPmap) designed to automatically assign new protein structures to existing evolutionary-based classification schemes, we identify a evolutionarily conserved domain (EDD) common to three different folds: mannose transporter EIIA domain (EIIA-man), dihydroxyacetone kinase (Dak), and DegV. Several lines of evidence support unification of these three folds into a single superfamily: statistically significant sequence similarity detected by PSI-BLAST; "closed structural grouping" using DALI Z-scores (each protein inside a group finds all other group members with scores higher than those to proteins outside the group) that includes only these proteins sharing a unique alpha-helical hairpin at the C-terminus and excludes all other proteins with similar topology; similar domain fusions connect Dak and DegV, and genomic neighborhood organizations connect Dak and EIIA-man. Finally, both Dak and EIIA-man perform similar phosphotransfer reactions, suggesting a phosphotransferase activity for the DegV-like family of proteins, whose function other than lipid binding revealed in the crystal structure remains unknown.

Background Inference of remote homology between proteins is very challenging and remains a prerogative of an expert. Thus a significant drawback to the use of evolutionary-based protein structure classifications is the difficulty in assigning new proteins to unique positions in the classification scheme with automatic methods. To address this issue, we have developed an algorithm to map protein domains to an existing structural classification scheme and have applied it to the SCOP database. Results The general strategy employed by this algorithm is to combine the results of several existing sequence and structure comparison tools applied to a query protein of known structure in order to find the homologs already classified in SCOP database and thus determine classification assignments. The algorithm is able to map domains within newly solved structures to the appropriate SCOP superfamily level with ~95% accuracy. Examples of correctly mapped remote homologs are discussed. The algorithm is also capable of identifying potential evolutionary relationships not specified in the SCOP database, thus helping to make it better. The strategy of the mapping algorithm is not limited to SCOP and can be applied to any other evolutionary-based classification scheme as well. SCOPmap is available for download. Conclusion The SCOPmap program is useful for assigning domains in newly solved structures to appropriate superfamilies and for identifying evolutionary links between different superfamilies.