Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding

Na+ binding to thrombin enhances the catalytic activity toward numerous synthetic and natural substrates. The bound Na+ is located in a solvent channel 16 A away from the catalytic triad, and connects with D189 in the S1 site through an intervening water molecule. Molecular modeling indicates that the G184K substitution in thrombin positions the protonated epsilon-amino group of the Lys side-chain to replace the bound Na+. Likewise, the G184R substitution positions the guanidinium group of the longer Arg side-chain to replace both the bound Na+ and the connecting water molecule to D189. We explored whether the G184K or G184R substitution would replace the bound Na+ and yield a thrombin derivative stabilized in the highly active fast form. Both the G184K and G184R mutants lost sensitivity to monovalent cations, as expected, but their activity toward a chromogenic substrate was compromised up to 200-fold as a result of impaired diffusion into the S1 site and decreased deacylation rate. Interestingly, both G184K and G184R substitutions compromised cleavage of procoagulant substrates fibrinogen and PAR1 more than that of the anticoagulant substrate protein C. These findings demonstrate that Na+ binding to thrombin is difficult to mimic functionally with residue side-chains, in analogy with results from other systems.     

Protease-activated receptor 4-like peptides bind to thrombin through an optimized interaction with the enzyme active site surface

Protease-activated receptor 4 (PAR4) is cleaved by thrombin at the R47-G48 peptide bond. Unlike PAR1, PAR4 does not contain a sequence readily predicted to interact with thrombin anion binding exosite-I. HPLC kinetic results on hydrolysis of PAR4 peptides (38-51 and 38-62) reveal that extending the sequence from the active site toward the exosite does not promote further binding interactions with thrombin. One-dimensional-proton line-broadening NMR indicates that the amino acids occupying the P(4)-P(1) positions of PAR4 (38-47), 44PAPR(47), come into direct contact with the thrombin surface. Less contact arises from the Leu43 at the P(5) position. Two-dimensional total correlation spectroscopy and two-dimensional transferred nuclear Overhauser effect spectroscropy studies on this complex reveal that Leu43 is flexible and can exhibit two conformational states. The binding mode observed for PAR4 peptides is similar to that of PAR1 peptides. PAR4 takes advantage of a distinctive sequence to optimize its interactions with the thrombin active site surface.     

Probing the S1/S1' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.     

Mechanism by which exosites promote the inhibition of blood coagulation proteases by heparin-activated antithrombin

Heparin activates the serpin, antithrombin, to inhibit its target blood-clotting proteases by generating new protease interaction exosites. To resolve the effects of these exosites on the initial Michaelis docking step and the subsequent acylation and conformational change steps of antithrombin-protease reactions, we compared the reactions of catalytically inactive S195A and active proteases with site-specific fluorophore-labeled antithrombins that allow monitoring of these reaction steps. Heparin bound to N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled antithrombins and accelerated the reactions of the labeled inhibitor with thrombin and factor Xa similar to wild type. Equilibrium binding of NBD-labeled antithrombins to S195A proteases showed that exosites generated by conformationally activating antithrombin with a heparin pentasaccharide enhanced the affinity of the serpin for S195A factor Xa minimally 100-fold. Moreover, additional bridging exosites provided by a hexadecasaccharide heparin activator enhanced antithrombin affinity for both S195A factor Xa and thrombin at least 1000-fold. Rapid kinetic studies showed that these exosite-mediated enhancements in Michaelis complex affinity resulted from increases in k(on) and decreases in k(off) and caused antithrombin-protease reactions to become diffusion-controlled. Competitive binding and kinetic studies with exosite mutant antithrombins showed that Tyr-253 was a critical mediator of exosite interactions with S195A factor Xa; that Glu-255, Glu-237, and Arg-399 made more modest contributions to these interactions; and that exosite interactions reduced k(off) for the Michaelis complex interaction. Together these results show that exosites generated by heparin activation of antithrombin function both to promote the formation of an initial antithrombin-protease Michaelis complex and to favor the subsequent acylation of this complex.     

Sodium ions as the effector of catalytic action of alpha-thrombin

A process of thrombin interaction with synthetic and natural substrates in the presence of Na+ ions has been analyzed in the survey. Molecular bases of this interaction have been presented, interrelation between the structure and function of thrombin has been noted; the nature of the unique site of its active centre which determines high thrombin affinity for the substrates and increase of its catalytic activity defined by the term of "specificity to univalent cations" have been considered in detail. Na+ ions play the role of allosteric effector in realization of two informational states of thrombin which penform, respectively, two fundamental and competing functions in the process of hemostasis. The molecular basis of the process of Na+ binding with thrombin is rather simple and depends only on the single site which importance for the enzyme function is marked by numerous investigations of a number of authors, and it is shown that Na(+)-binding site is distributed in the other zone of thrombin molecule as compared to exosites I and II, which do not take part in Na(+)-binding and allosteric transduction. Considerable attention was given to conformational conversions of a thrombin molecule caused by Na+ ions binding. It was shown that the transition slow <--> fast of the enzyme forms leads to formation of the ion pair Arg-187: Asp-222, optimal orientation of Asp-189 and Ser-195 for binding of substrates and considerable shift of the lateral chain Glu-192 determined by the disturbance of the lattice of water molecules which connects Na(+)-binding site with aminoacid Ser-195 of the active centre of the enzyme. New data have been presented which indicate that the changes in the lattice of water molecules and allosteric nucleus of Na(+)-binding site of the enzyme are the basic link of raising the affinity between the thrombin and substrate and mechanism of the enzyme activation by Na(+)-ions. The survey touches some problems of creation of allosteric inhibitors of thrombin which can take essential effect on Na(+)-binding site and favor stabilization of the anticoagulant slow-form of thrombin, and of enzyme rational mutants with selective specificity in respect of protein C which display effective and safe anticoagulant and antithrombotic effects in vivo.     

Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells

Activated protein C (APC) has endothelial barrier protective effects that require binding to endothelial protein C receptor (EPCR) and cleavage of protease activated receptor-1 (PAR1) and that may play a role in the anti-inflammatory action of APC. In this study we investigated whether protein C (PC) activation by thrombin on the endothelial cell surface may be linked to efficient protective signaling. To minimize direct thrombin effects on endothelial permeability we used the anticoagulant double mutant thrombin W215A/E217A (WE). Activation of PC by WE on the endothelial cell surface generated APC with high barrier protective activity. Comparable barrier protective effects by exogenous APC required a 4-fold higher concentration of APC. To demonstrate conclusively that protective effects in the presence of WE are mediated by APC generation and not direct signaling by WE, we used a PC variant with a substitution of the active site serine with alanine (PC S360A). Barrier protective effects of a low concentration of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. WE induced protective signaling only in the presence of wild type PC but not PC S360A and PAR1 cleavage was required for these protective effects. These data demonstrate that the endogenous PC activation pathway on the endothelial cell surface is mechanistically linked to PAR1-dependent autocrine barrier protective signaling by the generated APC. WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC.     

Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel beta-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits alpha-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 1.75 +/- 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50 = 20 microM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 microM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active site of alpha-thrombin.     

In silico analyses of substrate interactions with human serum paraoxonase 1

Human paraoxonase (HuPON1) is a serum enzyme that exhibits a broad spectrum of hydrolytic activities, including the hydrolysis of various organophosphates, esters, and recently identified lactone substrates. Despite intensive site-directed mutagenesis and other biological studies, the structural basis for the specificity of substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The results suggest that the active site of HuPON1 is characterized by two distinct binding regions: the hydrophobic binding site for arylesters/lactones, and the paraoxon binding site for phosphotriesters. The unique binding modes proposed for each type of substrate reveal a number of key residues governing substrate specificity. The polymorphic residue R/Q192 interacts with the leaving group of paraoxon, suggesting it plays an important role in the proper positioning of this substrate in the active site. MD simulations of the optimal binding complexes show that residue Y71 undergoes an "open-closed" conformational change upon ligand binding, and forms strong interactions with substrates. Further binding free energy calculations and residual decomposition give a more refined molecular view of the energetics and origin of HuPON1/substrate interactions. These studies provide a theoretical model of substrate binding and specificity associated with wild type and mutant forms of HuPON1, which can be applied in the rational design of HuPON1 variants as bioscavengers with enhanced catalytic activity.     

Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and beta-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with beta-arrestin 1 (betaarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas beta-arrestin 2 (betaarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by betaarr1 compared with betaarr2. In contrast, both beta-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated betaarr1 in an agonist-dependent manner, whereas betaarr2 association was virtually undetectable. Remarkably, betaarr1 also interacted with phosphorylation defective PAR1 mutant, whereas betaarr2 did not. Moreover, constitutively active beta-arrestin mutants, betaarr1 R169E and betaarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, beta-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine(2A) receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, beta-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.     

Allosteric changes in thrombin's activity produced by peptides corresponding to segments of natural inhibitors and substrates.

Acidic synthetic peptides corresponding to segments of several nonhomologous proteins (hirudin, residues 54-65; heparin cofactor II, residues 54-75; and fibrinogen, residues 410-427 of the gamma B-chain) inhibit thrombin's cleavage of fibrinogen without blocking the enzyme's active site. Here, we examined effects of these peptides on thrombin's cleavage of protein C and small peptides. Activation of protein C by thrombin in the absence of calcium was inhibited by all of the peptides. Maximal inhibition was 60%, and no greater inhibition was produced by higher peptide concentrations. This differed from progressive inhibition of protein C activation by increasing peptide concentrations in the presence of thrombomodulin and calcium. Potencies of the peptides were in the order hirudin-(54-65) greater than heparin cofactor II-(54-75) greater than gamma B-chain-(410-427). Sulfation of the tyrosine residue in hirudin-(54-65) increased its potency about 10-fold, similar to changes in anticlotting activity. The peptides were activators rather than inhibitors of the cleavage of small chromogenic substrates. In the presence of the peptides, the affinity of thrombin for the substrates S-2366 (pyro-Glu-Pro-Arg-4-nitroanilide), Chromozyme TH (tosyl-Gly-Pro-Arg-4-nitroanilide), and S-2251 (D-Val-Leu-Lys-4-nitroanilide) increased 1.5-2-fold with little change in the Vmax of substrate cleavage. Potencies of peptides in these allosteric effects on thrombin was in the same order as for their other effects. The similar actions of these nonhomologous peptides, which are believed to bind to thrombin's anion-binding exosite, suggest that binding of any peptide to this site exerts the same allosteric effect on thrombin's active site. Interactions of these peptides with thrombin may serve as models for regulation of thrombin's interactions with natural substrates and inhibitors.     

Crystal structure of the anticoagulant slow form of thrombin

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na(+)-free slow form. The slow form shows subtle differences compared with the currently available structures of the Na(+)-bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190-193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.     

Hirudin: amino-terminal residues play a major role in the interaction with thrombin

Amino acid substitutions within the amino-terminal 5 residues of the thrombin-specific inhibitor hirudin dramatically alter its ability to inhibit the thrombin-catalyzed hydrolysis of both a chromogenic substrate and fibrinogen. Replacing the highly conserved Tyr-3 residue with Trp or Phe increases hirudin's affinity for thrombin 3-6-fold (decreases the inhibition constant, Ki) whereas Thr results in a 450-fold increase in Ki. A more extensive modification involving deletion of the amino-terminal Val, and Tyr-3----Val, Thr-4----Gln, and Asp-5----Ile replacement, results in a large reduction in thrombin inhibitory activity corresponding to greater than a 10(7)-fold increase in Ki and a 10(3)-fold increase in IC50, using D-Phe-L-pipecolyl-Arg-p-nitroanilide (S-2238) and fibrinogen, respectively, as substrates. Kinetic analysis of these mutant proteins and synthetic peptide fragments and available structural information on thrombin and hirudin derived from protein crystallography and two-dimensional NMR studies indicate that the amino-terminal region of hirudin binds at the apolar binding/active site region of thrombin, with Tyr-3 occupying the S3 specificity site. The large effect of these modifications on hirudin activity suggests that alteration of the amino-terminal segment can destabilize the interaction of other regions of hirudin with thrombin.     

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.     

Site-directed mutagenesis of the beta subunit of tryptophan synthase from Salmonella typhimurium. Role of active site glutamic acid 350.

To investigate the functional role of glutamic acid 350 in the active site of the beta subunit of tryptophan synthase from Salmonella typhimurium, we have replaced this residue by glutamine or alanine by use of site-directed mutagenesis. The mutant alpha 2 beta 2 complexes were expressed, purified, crystallized, and characterized by spectroscopic and kinetic studies with several substrates. We find large alterations in the substrate and reaction specificity of each mutant form of the alpha 2 beta 2 complex. Since the two mutant enzymes are virtually inactive in reactions with L-serine but are active in reactions with beta-chloro-L-alanine, glutamic acid 350 may facilitate the beta-elimination of the weak hydroxyl leaving group of L-serine. The mutant alpha 2 beta 2 complexes are more active than the wild type enzyme in the beta-elimination reaction with beta-chloro-L-alanine. These enzymes are irreversibly inactivated by beta-chloro-L-alanine, whereas the wild type enzyme is not. These altered properties may result from a change in the conformation of the active site, from a change in the orientation of the coenzyme relative to active site residues, or from a change in the solvent accessibility of the active site. The alteration in the active site may enhance the release of amino acrylate from the Schiff base intermediate by hydrolysis or by transamination.     

X-ray snapshots of peptide processing in mutants of tricorn-interacting factor F1 from Thermoplasma acidophilum

The tricorn-interacting factor F1 of the archaeon Thermoplasma acidophilum cleaves small hydrophobic peptide products of the proteasome and tricorn protease. F1 mutants of the active site residues that are involved in substrate recognition and catalysis displayed distinct activity patterns toward fluorogenic test substrates. Crystal structures of the mutant proteins complexed with peptides Phe-Leu, Pro-Pro, or Pro-Leu-Gly-Gly showed interaction of glutamates 213 and 245 with the N termini of the peptides and defined the S1 and S1' sites and the role of the catalytic residues. Evidence was found for processive peptide cleavage in the N-to-C direction, whereby the P1' product is translocated into the S1 site. A functional interaction of F1 with the tricorn protease was observed with the inactive F1 mutant G37A. Moreover, small angle x-ray scattering measurements for tricorn and inhibited F1 have been interpreted as formation of transient and substrate-induced complexes.     

Site-directed mutagenesis of Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase: Lys-23 is essential for substrate binding

Chemical modification of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine), with pyridoxal 5'-phosphate suggested that Lys-22 (equivalent to Lys-23 of the Petunia hybrida enzyme) is a potential active site residue (Huynh, Q. K., Kishore, G. M., and Bild, G. S. (1988) J. Biol. Chem. 263, 735-739). To investigate the possible role of this residue in the reaction mechanism, we have used site-directed mutagenesis to replace Lys-23 of the P. hybrida enzyme with 3 other amino acid residues: Ala, Glu, and Arg. Analysis of these mutant enzymes indicates that of these only the Lys-23 to Arg mutant enzyme is active; the other two replacements (Ala and Glu) result in inactivation of the enzyme. Two of the mutant enzymes (Lys-23 to Arg and Ala) were purified to homogeneity and characterized. The purified Lys-23 to Arg mutant enzyme is less sensitive than the wild type enzyme to pyridoxal 5'-phosphate. It showed identical Km values for substrates and a 5-fold higher I50 value for glyphosate in comparison with those from the wild type enzyme. Binding studies using fluorescence measurements revealed that the substrate shikimate 3-phosphate and glyphosate were able to bind the purified Lys-23 to Arg mutant enzyme but not to the purified catalytically inactive Lys-23 to Ala mutant enzyme. The above results suggest that the cationic group at position 23 of the enzyme may play an important role in substrate binding.     

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.

We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.     

Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin.

NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

The Arabidopsis phosphatase PP2C49 negatively regulates salt tolerance through inhibition of AtHKT1;1

Type 2C protein phosphatases (PP2Cs) are the largest protein phosphatase family. PP2Cs dephosphorylate substrates for signaling in Arabidopsis, but the functions of most PP2Cs remain unknown. Here, we characterized PP2C49 (AT3G62260, a Group G PP2C), which regulates Na⁺ distribution under salt stress and is localized to the cytoplasm and nucleus. PP2C49 was highly expressed in root vascular tissues and its disruption enhanced plant tolerance to salt stress. Compared with wild type, the pp2c49 mutant contained more Na⁺ in roots but less Na⁺ in shoots and xylem sap, suggesting that PP2C49 regulates shoot Na⁺ extrusion. Reciprocal grafting revealed a root‐based mechanism underlying the salt tolerance of pp2c49. Systemic Na⁺ distribution largely depends on AtHKT1;1 and loss of function of AtHKT1;1 in the pp2c49 background overrode the salt tolerance of pp2c49, resulting in salt sensitivity. Furthermore, compared with plants overexpressing PP2C49 in the wild‐type background, plants overexpressing PP2C49 in the athtk1;1 mutant background were sensitive to salt, like the athtk1;1 mutants. Moreover, protein–protein interaction and two‐voltage clamping assays demonstrated that PP2C49 physically interacts with AtHKT1;1 and inhibits the Na⁺ permeability of AtHKT1;1. This study reveals that PP2C49 negatively regulates AtHKT1;1 activity and thus determines systemic Na⁺ allocation during salt stress.