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Query: UNIPROT:P06889 (Mol)
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The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.
J Mol Biol 1991 Sep 05
PMID:Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution. 192 Apr 11

A low molecular weight protein inhibitor of serine proteinases from Russet Burbank potato tubers, polypeptide chymotrypsin inhibitor-1 (PCI-1), has been crystallized in complex with Streptomyces griseus proteinase B (SGPB). The three-dimensional structure of the complex has been solved at 2.1 A resolution by the molecular replacement method and has been refined to a final R-factor (= sigma[[Fo[-[Fc[[/sigma[Fo[) of 0.142 (8.0 to 2.1 A resolution data). The reactive site bond of PCI-1 (Leu38I to Asn39I) is intact in the complex, and there is no significant distortion of the peptide from planarity. The distance between the active site serine O gamma of SGPB and the carbonyl carbon of the scissile bond of PCI-1 is 2.8 A (1 A = 0.1 nm). The inhibitor has little secondary structure, having a three-stranded antiparallel beta-sheet on the side opposite the reactive site and four beta-turns. PCI-1 has four disulphide bridges; these presumably take the place of extensive secondary structure in keeping the reactive site conformationally constrained. The pairing of the cystine residues, which had not been characterized chemically, is as follows: Cys3I to Cys40I, Cys6I to Cys24I, Cys7I to Cys36I, and Cys13I to Cys49I. The molecular structure of SGPB in the PCI-1 complex agrees closely with the structure of SGPB complexed with the third domain of the turkey ovomucoid inhibitor (OMTKY3). A least-squares overlap of all atoms in SGPB gives a root-mean-square difference of 0.37 A. One of the loops of SGPB (Ser35 to Gly40) differs in conformation in the two complexes by more than 2.0 A root-mean-square for the main-chain atoms. Thr39 displays the largest differences with the carbonyl carbon atom deviating by 3.6 A. This conformational alternative is a result of the differences in the molecular structures of the P'4 residues following the reactive site bonds of the two inhibitors. This displacement avoids a close contact (1.3 A) between the carbonyl oxygen of Ser38 of SGPB and Pro42I C beta of PCI-1. The solvent structure of the PCI-1-SGPB complex includes 179 waters, two sulphate or phosphate ions, and one calcium or potassium ion, which appears to play a role in crystal formation. The molecular structure of PCI-1 determined here has allowed the proposal of a model for the structure of a two-domain inhibitor from potatoes and tomatoes, inhibitor II.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Jan 05
PMID:Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from Russet Burbank potato tubers at 2.1 A resolution. 249 44

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].
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PMID:The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3). 397 87

An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated delta G contribution of the amino group from the BPTI residue Lys15 (9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal). In OMTKY3, different binding loop residues are known to affect differently the binding (delta delta G) to alpha-chymotrypsin and protease B; a good qualitative agreement was found between the calculated delta G(residue) estimates and the experimental delta delta G data (correlation coefficient 0.7). Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains).(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1993 Dec 05
PMID:Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures. 825 66

Streptomyces griseus protease B (SGPB) has primary specificity for large hydrophobic residues. The protease is secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity. We genetically substituted the P1 Leu at the promature junction of SGPB with Phe, Met, or Val and monitored expression levels in Escherichia coli. Substitution with Phe had no effect on active SGPB production; substitution with Met or Val abolished proteolytic activity. An E. coli expression library containing 29,952 possible SGPB mutants was constructed with variations at seven sites involved in conferring primary specificity. A rapid, visual screening strategy was used to detect active protease secretion. The expression library was screened, in conjunction with the different promature junction sequences, for those variants producing increased proteolytic activity. The sequences of the isolated mutant genes were determined; the substrate specificities and thermostabilities of the corresponding protease were investigated. Mutants isolated from the screen with the wild-type promature junction exhibited substrate specificities and thermostabilities similar to wild-type. The screen with Phe at the promature junction P1 site resulted in the isolation of mutant proteases with increased thermostabilities (up to an order of magnitude increase in half-life at 55 degrees C), while a protease with broad substrate specificity was isolated from Val screen. Proteases isolated from the screen with Met at the promature junction P1 site exhibited dramatic increases in activity towards a synthetic substrate with Met at P1 site. The results suggests that the substrate specificity of recombinant SGPB is constrained by the sequence of the promature junction; active protease production is dependent on the efficiency of the self-processive promature junction cleavage. With an efficient screening strategy, this relationship can be used to isolate catalytically active proteases with desired specificities engineered at the promature junction.
J Mol Biol 1996 Mar 29
PMID:Selection of Streptomyces griseus protease B mutants with desired alterations in primary specificity using a library screening strategy. 860 20

Binding constants for complexes of variants of the ovomucoid inhibitor domain 3 from turkey (OMTKY3) and Streptomyces griseus protease B (SGPB) have been computed. On the basis of the crystallographically determined structures of the complexes, continuum electrostatic calculations have been carried out to evaluate the electrostatic contribution to the binding energy. The hydrophobic component was computed based on the change in the solvent accessible surface area on complex formation. These two terms were combined linearly and the parameters for the protein dielectric, atomic solvation parameter and a constant term were derived using a multivariate fit to the observed binding energies. The resulting fit shows a high correlation with a multiple coefficient of determination of 0.79. This indicates that 79% of the variation in the observed binding energies is explained by the electrostatic and hydrophobic terms. The analysis results in a protein dielectric of 8.2 and an atomic solvation parameter of 30 cal/mol A2. As a test, these parameters were used to calculate the binding energies of complexes of chymotrypsin and of leukocyte elastase OMTKY3, as well as three other variants of OMTKY3 bound to SGPB. As these structures were not used for the multivariate fit, they serve as an independent check on the derived parameters. The calculated energies for the three new variants of OMTKY3 are in good agreement with the observed values. However, the binding energies of the other complexes are poorly predicted. This implies that the parameters that were obtained are not transferable. The possible causes for this lack of transferability are discussed.
J Mol Biol 1998 Dec 18
PMID:Computational analysis of the binding of P1 variants of domain 3 of turkey ovomucoid inhibitor to Streptomyces griseus protease B. 987 79

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.
J Mol Biol 2001 Jan 26
PMID:Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I. 1116 96

The construction and characterization of a novel, thermostable, peptide ligase are described. Three amino acid substitutions were introduced into the secreted bacterial protease Streptomyces griseus protease B (SGPB). Mutations were chosen on the basis of two separate observations: (i) that a single substitution of the nucleophilic serine (S195A) created an enzyme with significant peptide-ligation activity, albeit greatly reduced stability [(2000) Chem. Biol. 7, 163], and (ii) that a pair of substitutions in the substrate-binding pocket (T213L and F228H) greatly increased the thermostability of the wild-type enzyme [(1996) J. Mol. Biol. 257, 233]. The triple mutant, named streptoligase, was found to catalyze peptide ligation (aminolysis of both a thiobenzyl ester and a p-nitroanilide-activated peptide) efficiently in nondenaturing and denaturing conditions including SDS (0.5% w/v) and guanidine hydrochloride (4.0 M). Moreover, streptoligase exhibited a half-live for unfolding of 16.3 min at 55 degrees C in the absence of stabilizing substrates. The fraction of the streptoligase-catalyzed reaction that gave coupled product with the acceptor peptide FAASR-NH(2) was greater for the p-nitroanilide donor (Sc-AAPF-pNA) than for the benzyl thioester substrate (Sc-AAPF-SBn). These observations are consistent with ligation proceeding through an acyl-enzyme intermediate involving histidine-57. In the case of the thioester donor the triple mutant promotes the direct attack of water on the thioester carbonyl carbon, in addition to hydrolysis occurring at the stage of the acyl-enzyme intermediate. The strategy of multiple point mutations outlined in this study may provide a general means of converting enzymes with chymotrypsin-like protein folds into peptide ligases.
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PMID:Generation of a thermostable and denaturant-resistant peptide ligase. 1519 9

Alpha-lytic protease (alpha LP) and Streptomyces griseus protease B (SGPB) are two extracellular serine proteases whose folding is absolutely dependent on the existence of their companion pro regions. Moreover, the native states of these proteins are, at best, marginally stable, with the apparent stability resulting from being kinetically trapped in the native state by large barriers to unfolding. Here, in an effort to understand the physical properties that distinguish kinetically and thermodynamically stable proteins, we study the temperature-dependences of the folding and unfolding kinetics of alpha LP and SGPB without their pro regions, and compare their behavior to a comprehensive set of other proteins. For the folding activation thermodynamics, we find some remarkable universal behaviors in the thermodynamically stable proteins that are violated dramatically by alpha LP. Despite significant variations in deltaC(P,F)++, the maximal folding speed occurs within the narrow biological temperature range for all proteins, except for alpha LP, with its maximal folding speed shifted lower by 200 K. This implies evolutionary pressures on folding speed for typical proteins, but not for alpha LP. In addition, the folding free energy barrier in the biological temperature range for most proteins is predominantly enthalpic, but purely entropic for alpha LP. The unfolding of alpha LP and SGPB is distinguished by three properties: a remarkably large deltaC(P,U)++, a very high deltaG(U)++, and a maximum deltaG(u)++ at the optimal growth temperature for the organism. While other proteins display each of these traits to some approximation, the simultaneous optimization of all three occurs only in the kinetically stable proteins, and appears to be required to maximize their unfolding cooperativity, by suppressing local unfolding events, and slowing the rate of global unfolding. Together, these properties extend the lifetime of these enzymes in the highly proteolytic extracellular environment. Attaining such functional properties seems possible only through the gross perturbation of the folding thermodynamics, which in turn has required the co-evolution of pro regions as folding catalysts.
J Mol Biol 2005 Mar 25
PMID:Comprehensive analysis of protein folding activation thermodynamics reveals a universal behavior violated by kinetically stable proteases. 1574 Jul 46

Sequence-to-reactivity algorithms (SRAs) for proteins have the potential of being broadly applied in molecular design. Recently, Laskowski et al. have reported an additivity-based SRA that accurately predicts most of the standard free energy changes of association for variants of turkey ovomucoid third domain (OMTKY3) with six serine peptidases, one of which is streptogrisin B (commonly known as Streptomyces griseus peptidase B, SGPB). Non-additivity effects for residues 18I and 32I, and for residues 20I and 32I of OMTKY3 occurred when the associations with SGPB were predicted using the SRA. To elucidate precisely the mechanics of these non-additivity effects in structural terms, we have determined the crystal structures of the unbound OMTKY3 (with Gly32I as in the wild-type amino acid sequence) at a resolution of 1.16 A, the unbound Ala32I variant of OMTKY3 at a resolution of 1.23 A, and the SGPB:OMTKY3-Ala32I complex (equilibrium association constant K(a)=7.1x10(9) M(-1) at 21(+/-2) C degrees, pH 8.3) at a resolution of 1.70 A. Extensive comparisons with the crystal structure of the unbound OMTKY3 confirm our understanding of some previously addressed non-additivity effects. Unexpectedly, SGPB and OMTKY3-Ala32I form a 1:2 complex in the crystal. Comparison with the SGPB:OMTKY3 complex shows a conformational change in the SGPB:OMTKY3-Ala32I complex, resulting from a hinged rigid-body rotation of the inhibitor caused by the steric hindrance between the methyl group of Ala32IA of the inhibitor and Pro192BE of the peptidase. This perturbs the interactions among residues 18I, 20I, 32I and 36I of the inhibitor, probably resulting in the above non-additivity effects. This conformational change also introduces residue 10I as an additional hyper-variable contact residue to the SRA.
J Mol Biol 2007 Mar 23
PMID:Structural insights into the non-additivity effects in the sequence-to-reactivity algorithm for serine peptidases and their inhibitors. 1726 86


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