FireDB--a database of functionally important residues from proteins of known structure.
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ABSTRACT:
SUBMITTER: Lopez G
PROVIDER: S-EPMC1716728 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MotivationMutating residues into alanine (alanine scanning) is one of the fastest experimental means of probing hypotheses about protein function. Alanine scans can reveal functional hot spots, i.e. residues that alter function upon mutation. In vitro mutagenesis is cumbersome and costly: probing all residues in a protein is typically as impossible as substituting by all non-native amino acids. In contrast, such exhaustive mutagenesis is feasible in silico.ResultsPreviously, we developed SNAP to predict functional changes due to non-synonymous single nucleotide polymorphisms. Here, we applied SNAP to all experimental mutations in the ASEdb database of alanine scans; we identi.ed 70% of the hot spots (>or=1 kCal/mol change in binding energy); more severe changes were predicted more accurately. Encouraged, we carried out a complete all-against-all in silico mutagenesis for human glucokinase. Many of the residues predicted as functionally important have indeed been con.rmed in the literature, others await experimental veri.cation, and our method is ready to aid in the design of in vitro mutagenesis.AvailabilityASEdb and glucokinase scores are available at http://www.rostlab.org/services/SNAP. For submissions of large/whole proteins for processing please contact the author.
| S-EPMC2597370 | biostudies-literature
Project description:Thrombopoietin (TPO) is a haematopoietic growth factor responsible for megakaryocyte progenitor proliferation and differentiation. It belongs to the four-helix-bundle cytokine family and exerts its biological effects through binding to a specific receptor, c-Mpl. With the use of site-directed mutagenesis we have generated 20 TPO mutants. Each of the TPO mutants was produced in a eukaryotic expression system and the mutants' ability to induce the proliferation of factor-dependent c-Mpl-expressing megakaryoblastic M-O7e cells was compared with that of wild-type TPO. Among the mutations studied, 10 lead to a significant decrease in TPO bioactivity. Of these ten residues, three are located in helix A of the protein (Arg10, Lys14 and Arg17) and four in helix D (His133, Gln132, Lys138 and Phe141), indicating that in TPO, as in other cytokines, these two helices are important for functional cytokine/receptor interactions. Surprisingly, mutant Arg10-->Ala (R10A) lacked any proliferative activity, despite the fact that this mutation was recently reported to have no effect on TPO/c-Mpl binding in a TPO phage ELISA [Pearce, Potts, Presta, Bald, Fendly and Wells (1997) J. Biol. Chem. 272, 20595-20602]. The lack of M-O7e proliferation is probably due to an inability of R10A mutant to promote receptor dimerization and thus receptor activation. Moreover we found that the Arg10 and Arg17 residues of TPO seem to be specific determinants for TPO/c-Mpl recognition. We also demonstrate that the O-glycosylation site located at position 110 of TPO is not necessary for the bioactivity of the cytokine.
| S-EPMC1219638 | biostudies-other
Project description:High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C(β) atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C(β) atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.
| S-EPMC4093959 | biostudies-literature
Project description:BackgroundWell-performing automated protein function recognition approaches usually comprise several complementary techniques. Beside constructing better consensus, their predictive power can be improved by either adding or refining independent modules that explore orthogonal features of proteins. In this work, we demonstrated how the exploration of global atomic distributions can be used to indicate functionally important residues.ResultsUsing a set of carefully selected globular proteins, we parametrized continuous probability density functions describing preferred central distances of individual protein atoms. Relative preferred burials were estimated using mixture models of radial density functions dependent on the amino acid composition of a protein under consideration. The unexpectedness of extraordinary locations of atoms was evaluated in the information-theoretic manner and used directly for the identification of key amino acids. In the validation study, we tested capabilities of a tool built upon our approach, called SurpResi, by searching for binding sites interacting with ligands. The tool indicated multiple candidate sites achieving success rates comparable to several geometric methods. We also showed that the unexpectedness is a property of regions involved in protein-protein interactions, and thus can be used for the ranking of protein docking predictions. The computational approach implemented in this work is freely available via a Web interface at http://www.bioinformatics.org/surpresi.ConclusionsProbabilistic analysis of atomic central distances in globular proteins is capable of capturing distinct orientational preferences of amino acids as resulting from different sizes, charges and hydrophobic characters of their side chains. When idealized spatial preferences can be inferred from the sole amino acid composition of a protein, residues located in hydrophobically unfavorable environments can be easily detected. Such residues turn out to be often directly involved in binding ligands or interfacing with other proteins.
| S-EPMC3188475 | biostudies-literature
Project description:Lack of crystal structure data of folate binding proteins has left so many questions unanswered (for example, important residues in active site, binding domain, important amino acid residues involved in interactions between ligand and receptor). With sequence alignment and PROSITE motif identification, we attempted to answer evolutionarily significant residues that are of functional importance for ligand binding and that form catalytic sites. We have analyzed 46 different FRs and FBP sequences of various organisms obtained from Genbank. Multiple sequence alignment identified 44 highly conserved identical amino acid residues with 10 cysteine residues and 12 motifs including ECSPNLGPW (which might help in the structural stability of FR).
| S-EPMC2255074 | biostudies-literature
Project description:Here we present firestar, an expert system for predicting ligand-binding residues in protein structures. The server provides a method for extrapolating from the large inventory of functionally important residues organized in the FireDB database and adds information about the local conservation of potential-binding residues. The interface allows users to make queries by protein sequence or structure. The user can access pairwise and multiple alignments with structures that have relevant functionally important binding sites. The results are presented in a series of easy to read displays that allow users to compare binding residue conservation across homologous proteins. The binding site residues can also be viewed with molecular visualization tools. One feature of firestar is that it can be used to evaluate the biological relevance of small molecule ligands present in PDB structures. With the server it is easy to discern whether small molecule binding is conserved in homologous structures. We found this facility particularly useful during the recent assessment of CASP7 function prediction.http://firedb.bioinfo.cnio.es/Php/FireStar.php.
| S-EPMC1933227 | biostudies-literature
Project description:The rapidly increasing volume of sequence and structure information available for proteins poses the daunting task of determining their functional importance. Computational methods can prove to be very useful in understanding and characterizing the biochemical and evolutionary information contained in this wealth of data, particularly at functionally important sites. Therefore, we perform a detailed survey of compositional and evolutionary constraints at the molecular and biological function level for a large set of known functionally important sites extracted from a wide range of protein families. We compare the degree of conservation across different functional categories and provide detailed statistical insight to decipher the varying evolutionary constraints at functionally important sites. The compositional and evolutionary information at functionally important sites has been compiled into a library of functional templates. We developed a module that predicts functionally important columns (FIC) of an alignment based on the detection of a significant "template match score" to a library template. Our template match score measures an alignment column's similarity to a library template and combines a term explicitly representing a column's residue composition with various evolutionary conservation scores (information content and position-specific scoring matrix-derived statistics). Our benchmarking studies show good sensitivity/specificity for the prediction of functional sites and high accuracy in attributing correct molecular function type to the predicted sites. This prediction method is based on information derived from homologous sequences and no structural information is required. Therefore, this method could be extremely useful for large-scale functional annotation.
| S-EPMC2222836 | biostudies-literature
Project description:Permeases belonging to the equilibrative nucleoside transporter family promote uptake of nucleosides and/or nucleobases into a wide range of eukaryotes and mediate the uptake of a variety of drugs used in the treatment of cancer, heart disease, AIDS, and parasitic infections. No experimental three-dimensional structure exists for any of these permeases, and they are not present in prokaryotes, the source of many membrane proteins used in crystal structure determination. To generate a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani was modeled using ab initio computation. Site-directed mutations that strongly impair transport or that alter substrate specificity map to the central pore of the ab initio model, whereas mutations that have less pronounced phenotypes map to peripheral positions. The model suggests that aromatic residues present in transmembrane helices 1, 2, and 7 may interact to form an extracellular gate that closes the permeation pathway in the inward oriented conformation. Mutation of two of these three residues abrogated transport activity, consistent with the prediction of the model. The ab initio model is similar to one derived previously using threading analysis, a distinct computational approach, supporting the overall accuracy of both models. However, significant differences in helix orientation and residue position between the two models are apparent, and the mutagenesis data suggest that the ab initio model represents an improvement regarding structural details over the threading model. The putative gating interaction may also help explain differences in substrate specificity between members of this family.
| S-EPMC2707223 | biostudies-literature
Project description:Lignostilbene-α,β-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (k cat app = 1.00 ± 0.04 × 106 m-1 s-1). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (Kic = 6 ± 1 μm, Kiu = 24 ± 4 μm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed β-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo A structure of an LsdA·phenylazophenol complex identified Phe59, Tyr101, and Lys134 as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe59 and Tyr101 substitutions with His and Phe, respectively, reduced LsdA activity (k cat app) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys134 plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.
| S-EPMC6721942 | biostudies-literature
Project description:Powered by ATP hydrolysis, P(IB) -ATPases drive the energetically uphill transport of transition metals. These high affinity pumps are essential for heavy metal detoxification and delivery of metal cofactors to specific cellular compartments. Amino acid sequence alignment of the trans-membrane (TM) helices of P(IB)-ATPases reveals a high degree of conservation, with ∼ 60-70 fully conserved positions. Of these conserved positions, 6-7 were previously identified to be important for transport. However, the functional importance of the majority of the conserved TM residues remains unclear. To investigate the role of conserved TM residues of P(IB)-ATPases we conducted an extensive mutagenesis study of a Zn(2+)/Cd(2+) P(IB)-ATPase from Rhizobium radiobacter (rrZntA) and seven other P(IB)-ATPases. Of the 38 conserved positions tested, 24 had small effects on metal tolerance. Fourteen mutations compromised in vivo metal tolerance and in vitro metal-stimulated ATPase activity. Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function. We tentatively propose that the distribution of the mutable and immutable residues marks a possible trans-membrane metal translocation pathway. In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).
| S-EPMC4285229 | biostudies-literature