Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural determinants of specificity in the cysteine protease cruzain.

ABSTRACT: The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X-ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl-Arg-Ala-fluoromethyl ketone and benzoyl-Tyr-Ala-fluoromethyl ketone at 2.2 and 2.1 A, respectively, show a pH-dependent specificity switch. Glu 205 adjusts to restructure the S2 specificity pocket, conferring right binding to both hydrophobic and basic residues. Kinetic analysis of activated peptide substrates shows that substrates placing hydrophobic residues in the specificity pocket are cleaved at a broader pH range than hydrophilic substrates. These results demonstrate how cruzain binds both basic and hydrophobic residues and could be important for in vivo regulation of cruzain activity.

SUBMITTER: Gillmor SA

PROVIDER: S-EPMC2143760 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Structural determinants of specificity in the cysteine protease cruzain.

Gillmor S A SA Craik C S CS Fletterick R J RJ

Protein science : a publication of the Protein Society 19970801 8

The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X-ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl-Arg-Ala-fluoromethyl ketone and benzoyl-Tyr-Ala-fluoromethyl ketone at 2.2 and 2.1 A, respectively, show a pH-dependent specificity switch. Glu 205 adjusts to restructure the S2 speci ...[more]

PMID: 9260273

Similar Datasets

Structural determinants of tobacco vein mottling virus protease substrate specificity.

Project description:Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (?20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ?10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

| S-EPMC3005794 | biostudies-literature

Structural Determinants of Substrate Specificity of SplF Protease from Staphylococcus aureus.

Project description:Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.

| S-EPMC7926377 | biostudies-literature

Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus.

Project description:Cavally virus (CavV) is a mosquito-borne plus-strand RNA virus in the family Mesoniviridae (order Nidovirales). We present X-ray structures for the CavV 3C-like protease (3CLpro), as a free enzyme and in complex with a peptide aldehyde inhibitor mimicking the P4-to-P1 residues of a natural substrate. The 3CLpro structure (refined to 1.94 Å) shows that the protein forms dimers. The monomers are comprised of N-terminal domains I and II, which adopt a chymotrypsin-like fold, and a C-terminal α-helical domain III. The catalytic Cys-His dyad is assisted by a complex network of interactions involving a water molecule that mediates polar contacts between the catalytic His and a conserved Asp located in the domain II-III junction and is suitably positioned to stabilize the developing positive charge of the catalytic His in the transition state during catalysis. The study also reveals the structural basis for the distinct P2 Asn-specific substrate-binding pocket of mesonivirus 3CLpros.

| S-EPMC7111312 | biostudies-literature

Structure determinants defining the specificity of papain-like cysteine proteases.

Project description:Papain-like cysteine proteases are widely expressed enzymes that mostly regulate protein turnover in the acidic conditions of lysosomes. However, in the last twenty years, these proteases have been evidenced to exert specific functions within different organelles as well as outside the cell. The most studied proteases of this family are human cysteine cathepsins involved both in physiological and pathological processes. The specificity of each protease to its substrates is mostly defined by the structure of the binding cleft. Different patterns of amino acid motif in this area determine the interaction between the protease and the ligands. Moreover, this specificity can be altered under the specific media conditions and in case other proteins are present. Understanding how this network works would allow researchers to design the diagnostic selective probes and therapeutic inhibitors. Moreover, this knowledge might serve as a key for redesigning and de novo engineering of the proteases for a wide range of applications.

| S-EPMC9712828 | biostudies-literature

Structural and kinetic determinants of protease substrates.

Project description:Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in alpha-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.

| S-EPMC4042863 | biostudies-literature

Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity-A Structural and Biochemical Approach.

Project description:Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity-I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity.

| S-EPMC4924875 | biostudies-literature

Structural and energetic determinants of tyrosylprotein sulfotransferase sulfation specificity.

Project description:Tyrosine sulfation is a type of post-translational modification (PTM) catalyzed by tyrosylprotein sulfotransferases (TPST). The modification plays a crucial role in mediating protein-protein interactions in many biologically important processes. There is no well-defined sequence motif for TPST sulfation, and the underlying determinants of TPST sulfation specificity remains elusive. Here, we perform molecular modeling to uncover the structural and energetic determinants of TPST sulfation specificity.We estimate the binding affinities between TPST and peptides around tyrosines of both sulfated and non-sulfated proteins to differentiate them. We find that better differentiation is achieved after including energy costs associated with local unfolding of the tyrosine-containing peptide in a host protein, which depends on both the peptide's secondary structures and solvent accessibility. Local unfolding renders buried peptide-with ordered structures-thermodynamically available for TPST binding. Our results suggest that both thermodynamic availability of the peptide and its binding affinity to the enzyme are important for TPST sulfation specificity, and their interplay results into great variations in sequences and structures of sulfated peptides. We expect our method to be useful in predicting potential sulfation sites and transferable to other TPST variants. Our study may also shed light on other PTM systems without well-defined sequence and structural specificities.All the data and scripts used in the work are available at http://dlab.clemson.edu/research/Sulfation.

| S-EPMC4155453 | biostudies-literature

Inferring joint sequence-structural determinants of protein functional specificity.

Project description:Residues responsible for allostery, cooperativity, and other subtle but functionally important interactions remain difficult to detect. To aid such detection, we employ statistical inference based on the assumption that residues distinguishing a protein subgroup from evolutionarily divergent subgroups often constitute an interacting functional network. We identify such networks with the aid of two measures of statistical significance. One measure aids identification of divergent subgroups based on distinguishing residue patterns. For each subgroup, a second measure identifies structural interactions involving pattern residues. Such interactions are derived either from atomic coordinates or from Direct Coupling Analysis scores, used as surrogates for structural distances. Applying this approach to N-acetyltransferases, P-loop GTPases, RNA helicases, synaptojanin-superfamily phosphatases and nucleases, and thymine/uracil DNA glycosylases yielded results congruent with biochemical understanding of these proteins, and also revealed striking sequence-structural features overlooked by other methods. These and similar analyses can aid the design of drugs targeting allosteric sites.

| S-EPMC5770160 | biostudies-literature

Molecular determinants of the substrate specificity of the complement-initiating protease, C1r.

Project description:The serine protease, C1r, initiates activation of the classical pathway of complement, which is a crucial innate defense mechanism against pathogens and altered-self cells. C1r both autoactivates and subsequently cleaves and activates C1s. Because complement is implicated in many inflammatory diseases, an understanding of the interaction between C1r and its target substrates is required for the design of effective inhibitors of complement activation. Examination of the active site specificity of C1r using phage library technology revealed clear specificity for Gln at P2 and Ile at P1', which are found in these positions in physiological substrates of C1r. Removal of one or both of the Gln at P2 and Ile at P1' in the C1s substrate reduced the rate of C1r activation. Substituting a Gln residue into the P2 of the activation site of MASP-3, a protein with similar domain structure to C1s that is not normally cleaved by C1r, enabled efficient activation of this enzyme. Molecular dynamics simulations and structural modeling of the interaction of the C1s activation peptide with the active site of C1r revealed the molecular mechanisms that particularly underpin the specificity of the enzyme for the P2 Gln residue. The complement control protein domains of C1r also made important contributions to efficient activation of C1s by this enzyme, indicating that exosite interactions were also important. These data show that C1r specificity is well suited to its cleavage targets and that efficient cleavage of C1s is achieved through both active site and exosite contributions.

| S-EPMC3668718 | biostudies-literature

Computational study on substrate specificity of a novel cysteine protease 1 precursor from Zea mays.

Project description:Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand-enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.

| S-EPMC4100162 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data