E Di Cera

..Here we review the molecular basis of thrombin allostery as recently emerged from mutagenesis and structural studies. The role of Na+ in blood coagulation and the evolution of serine proteases are also discussed...

..That enables a simple classification of these functionally diverse enzymes and reveals unanticipated connections with ion transporters...

..The molecular mechanism of thrombin allostery is a remarkable example of long-range communication that offers a paradigm for many other biological systems...

..A new paradigm emerges where two forms of the protease, E* and E, are in allosteric equilibrium and determine biological activity and specificity...

..Thrombin mutants with selectively compromised activity toward fibrinogen and PAR1 are effective in vivo as anticoagulant and antithrombotic agents...

..Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes...

..Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors...

..We also draw attention to a functional polarity that exists in the serine protease fold, which sheds light on the structural linkages between the active site and exosites...

..All vitamin K-dependent proteases and some complement factors are subject to the Na(+)-dependent regulation discovered for thrombin. Na+ is therefore a key factor in the activation of zymogens in the coagulation and complement systems...

..Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation...

..It is now possible to extend this strategy to the study of other interactions of thrombin, as well as to related serine proteases...

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..Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na(+) activation is present in all Na(+)-activated trypsin-like proteases...

..The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general...

..These findings demonstrate that Na+ binding to thrombin is difficult to mimic functionally with residue side-chains, in analogy with results from other systems...

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..3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies...

..5 kcal/mol but < 3.0 kcal/mol. These results shed light on a basic aspect of the enzyme-substrate interaction in the entire family of trypsin-like serine proteases...

..These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215...

..The conversion, however, does not result into a K+-activated enzyme...

..This form, named S(*), corresponds to the inactive Na(+)-free slow form identified by early kinetic studies. A simple model of thrombin allostery that incorporates the contribution of S(*) is proposed...

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..In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation...

..From this analysis, M+ complexation has the potential to be an efficient regulator of enzyme catalysis and stability and offers novel strategies for protein engineering to improve enzyme function...

..The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na(+) activation is interwoven with substrate selectivity in the trypsin scaffold...

..The functional versatility of thrombin owes much to its evolutionary origin and results from structural determinants and mechanisms that can be exploited by pharmacologic intervention...

..The environment of W215 influences differentially three physiologically important interactions of thrombin, a feature that should assist in the separate study of each of these functions in vivo...

..These findings have broad implications in the rational design of enzymes with enhanced catalytic properties...

..A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains...

..The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties...

..Finally, the Ala substitution of two negatively charged residues in exosite II, Asp(100) and Asp(178), is found unexpectedly to significantly increase thrombomodulin binding...

..The crystal structure of the thrombin-PAR1 complex, solved at 2.2-A resolution, reveals the details of this long-range allosteric communication in terms of a network of polar interactions...

..A mutation in Ea predicted to introduce Na(+) binding results in apparently increased activity with ventralization of the embryo, an effect not observed with wild-type Ea mRNA...

..Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo...

..These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations...

..The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response...

..These findings reinforce the crucial role of the Asp222:Arg187 ion-pair in stabilizing the fast form of thrombin...

..Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general...

..This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl- binding to fibrin...

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..The E* --> E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway...

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..Extensive similarities suggest that these cascades were built by adding enzymes from the bottom of the cascade up and from similar macromolecular building blocks...

..The three-dimensional models suggest a possible pattern of recognition by thrombin that applies generally to other natural substrates...

..The S214C mutant is catalytically inactive, which suggests that during evolution the TCN-->AGY codon transitions for Ser(214) occurred through Thr intermediates...

..Mutations of Trp(215) provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications...

..The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates...

..These findings demonstrate that inactive mutants of enzymes make it possible to dissect the equilibrium components linked to substrate binding and complement information on the kinetic properties of the wild type...

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..Cumulatively, these experiments demonstrate that Mg2+ can bind to the sensing domain of PhoQ and establish the presence of distinct binding sites for Mg2+ and Ca2+ in the PhoQ protein...

..These findings provide a framework for deeper investigation into the evolutionary forces that shaped each peptidase family and a roadmap to develop a timeline for their expansion as an interconnected system...

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..Compound 5f was selective for inhibiting chymase versus eight serine proteases. Compound 6h was orally bioavailable in rats (F=39%), and orally efficacious in a hamster model of inflammation...

..These findings are consistent with functional data and broaden our understanding of how thrombin interacts with fibrinogen at the molecular level...

..Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex...

..The aim of this study was to delineate the molecular mechanism of direct WE-platelet interactions under static and shear conditions...

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..These results show that engineered thrombin derivatives that selectively activate protein C may represent useful therapeutic agents for the treatment of thrombotic disorders...

..Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity...

..Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to gamma' chains...

..WE may have powerful protective effects in systemic inflammation through signaling by the endogenously generated APC...

..These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease...