Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin

Braun, Paul; Dennis, S. M.; Hofsteenge, Jan; Stone, Stuart R.

doi:10.1021/bi00417a048

Cited by 187 publications

(198 citation statements)

References 40 publications

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“…However, mutation of this residue perturbs the free energy of hirudin binding to the fast form by only 0.4 kcal/mol. This finding is in agreement with mutagenesis studies of hirudin that have reported only a modest perturbation of hirudin affinity for the H51Q mutant (20). As to D221 and D222, these residues make no direct contacts with hirudin and therefore the effect seen upon mutation is indirect and mediated by a perturbation of the slow <--fast equilibrium (7).…”

Section: U [supporting

confidence: 90%

Identification of residues linked to the slow-->fast transition of thrombin.

Guinto

Vindigni

Ayala

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Residues energetically linked to the allosteric transition of thrombin from its anticoagulant slow form to the procoagulant fast form have been identified by sitedirected mutagenesis. The energetics of recognition by the two forms of the enzyme were probed by using a synthetic chromogenic substrate, fibrinogen, and hirudin. The thrombin residues E39, W60d, E192, D221, and D222 are linked to the slow --fast transition and are part of an "allosteric core" through which events originating at the Na+ binding loop propagate to other regions of the enzyme. The thrombin residues Y76, W96, W148, and R173 lie at the periphery of the allosteric core, affect recognition of fibrinogen and hirudin to the same extent in both forms, and are not linked to the slow -> fast transition.Thrombin is an allosteric enzyme existing in two forms, slow and fast (1), that have been targeted toward two important and opposite roles (2). The slow form is anticoagulant because it activates more specifically protein C, a potent inhibitor of blood coagulation. The fast form is procoagulant because it cleaves fibrinogen with higher specificity and promotes formation of a blood clot. Much of the control of thrombin function in hemostasis can be reduced to the control of the allosteric equilibrium between the slow and fast forms. Identification of the structural components that are energetically linked to the slow -> fast transition is therefore crucial for unraveling structure-function relationships and may eventually lead to innovative strategies for control of thrombin function in vivo.Identification of residues energetically linked to the allosteric transitions of thrombin is based on the properties of the following thermodynamic cycle ( The slow (S) and fast (F) forms bind a ligand (L) with a standard free energy AGs and AGf, while AGO and AG1 reflect the standard free energy changes for switching from the slow to the fast form in the absence and presence of the ligand. The coupling free energy in the cycle is given by AGc= AGf-AGs = AG'-AGO [2] and measures the difference in binding free energy between the fast and slow forms. The value of AGc is negative if the fast form binds with higher affinity and is positive otherwise.Residues energetically linked to the slow -* fast transition can be mapped from the effect of their mutation on the value of AGc.The foregoing approach developed for the study of recognition processes in cooperative systems (4) bears conceptually on recent analyses of mutational effects in proteins (5) and is also amenable of extension to the analysis of substrate binding in the transition state. In this case, the vertical edges in the thermodynamic cycle 1 represent the free energies of formation of the transition state in the slow and fast forms. The coupling free energy in the transition state is (4) AG* =,AG' -AGO = -RTln (ka/K) K) 3 and depends on the specificity constants of the fast and slow forms. Residues energetically linked to the slow --fast transition in the transition state can be mapped from the eff...

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Section: U [supporting

confidence: 90%

Identification of residues linked to the slow-->fast transition of thrombin.

Guinto

Vindigni

Ayala

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…A number of mutants have been made in the C-terminal tail of hirudin to investigate its interactions with thrombin (Braun et al, 1988;Stone et al, 1989;Betz et al, 1991a,b;Yue et al, 1992). These are summarized in Table 2.…”

Section: Discussionmentioning

confidence: 99%

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

et al. 1993

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The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin"-65 (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-PhePro-Arg-Pr~-(Gly)~-hirudin~~-~' (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower Ki than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (Pl-Pl') seems to be cleaved.

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“…The crystal structures of thrombin (Bode et al, 1989(Bode et al, , 1992 and its complexes with the polypeptide inhibitor hirudin (Kinemage 1; Griitter et al, 1990;Rydel et al, 1990Rydel et al, , 1991 cleft of thrombin, the C-terminal region binds to a positively charged surface groove called the fibrinogen-recognition exosite. The C-terminal region of recombinant hirudin (rhir) between residues 55 and 65 contains five negatively charged residues, and results from studies using site-directed mutagenesis indicate that each of these negatively charged residues (Asp 55: Glu 57: Glu 58: Glu 61', and Glu 62') contributes to the stabilization of the complex (Braun et al, 1988a;Betz et al, 1991). In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al, 1989). However, although protein engineering studies indicate that each of the negatively charged residues in the C-terminal region of hirudin makes about the same contribution to binding energy (Braun et al, 1988a;Stone et al, 1989;Betz et al, 1991), 727 …”

mentioning

confidence: 99%

“…The C-terminal region of recombinant hirudin (rhir) between residues 55 and 65 contains five negatively charged residues, and results from studies using site-directed mutagenesis indicate that each of these negatively charged residues (Asp 55: Glu 57: Glu 58: Glu 61', and Glu 62') contributes to the stabilization of the complex (Braun et al, 1988a;Betz et al, 1991). In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al, 1989).…”

mentioning

confidence: 99%

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Electrostatic interactions in the association of proteins: An analysis of the thrombin–hirudin complex

et al. 1992

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The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finitedifference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.Keywords: electrostatic interactions; hirudin; protein-protein interactions; thrombin Thrombin is a serine protease that plays a central role in blood coagulation. It cleaves fibrinogen to yield fibrin monomers that form the basis of the blood clot. In addition, thrombin activates a number of other proteins involved in coagulation. Thrombin can be distinguished from other serine proteases, such as trypsin, in that it achieves its specificity by using binding sites, called exosites, that are distant from the catalytic center (Fenton, 1988). The crystal structures of thrombin (Bode et al., 1989(Bode et al., , 1992 and its complexes with the polypeptide inhibitor hirudin (Kinemage 1; Griitter et al., 1990;Rydel et al., 1990Rydel et al., , 1991 cleft of thrombin, the C-terminal region binds to a positively charged surface groove called the fibrinogen-recognition exosite. The C-terminal region of recombinant hirudin (rhir) between residues 55 and 65 contains five negatively charged residues, and results from studies using site-directed mutagenesis indicate that each of these negatively charged residues (Asp 55: Glu 57: Glu 58: Glu 61', and Glu 62') contributes to the stabilization of the complex (Braun et al., 1988a;Betz et al., 1991). In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al., 1989). However, although protein engineering studies indicate that each of the negatively char...

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Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin

Cited by 187 publications

References 40 publications

Identification of residues linked to the slow-->fast transition of thrombin.

Identification of residues linked to the slow-->fast transition of thrombin.

Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

Electrostatic interactions in the association of proteins: An analysis of the thrombin–hirudin complex

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