Role of the A chain in thrombin function

Papaconstantinou, M. E.; Bah, Alaji; Di, Enrico

doi:10.1007/s00018-008-8179-y

Cited by 28 publications

(40 citation statements)

References 25 publications

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“…In the case of wild-type, the value of k cat /K m for cleavage of fibrinogen or activation of PAR1 is Ͼ100-fold higher than that for activation of protein C in the presence of thrombomodulin (Table 1). A general trend gleaned from the plot is that an Ala mutation of the thrombin scaffold tends to perturb recognition of fibrinogen and PAR1 more than protein C. These findings extend and complement previous mutagenesis studies from our laboratory (10,(15)(16)(17)(18)(19)(20)(21)(22)(23) and others (25).…”

Section: Methodssupporting

confidence: 83%

“…Most of the 97 Ala mutants of thrombin were reported in previous studies (10,(15)(16)(17)(18)(19)(20)(21)(22)(23). All mutants of Trp 215 , except W215M and W215T (see below), were expressed in baby hamster kidney cells and activated overnight at 25°C with a mixture of a 1:30 dilution of crude venom from Echis carinatus and 1:100 dilution of venom from Agkistrodon acutus (Sigma) in the presence of 10 mM benzamidine and 10 mM CaCl 2 .…”

Section: Methodsmentioning

confidence: 99%

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Engineering Thrombin for Selective Specificity toward Protein C and PAR1

Marino

Pelc

Vogt

et al. 2010

Journal of Biological Chemistry

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Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp 215 as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp 215 produces constructs featuring k cat /K m values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and protein C that span five orders of magnitude. Importantly, the effect of Trp 215 replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp 215 provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications.Engineering protease specificity remains an issue of considerable interest and importance (1). Within the same fold, trypsins prefer Arg/Lys side chains at the P1 position (2) of substrate but chymotrypsins prefer Phe/Tyr/Trp residues (3-6). Primary specificity, defined by the nature of the P1 residue, is not the sole determinant of protease function. Many trypsin-like proteases show substrate preference that extends beyond the P1 position of substrate and is dictated by interactions with other structural domains not in contact with the primary specificity pocket. Indeed, inspection of consensus sequences recognized by thrombin for its three primary physiological targets, i.e. the procoagulant substrate fibrinogen, the prothrombotic protease-activated receptor 1 (PAR1) 2 and the anticoagulant substrate protein C, reveals an Arg residue at the P1 position in all cases (7). Hence, selection among these targets must depend on interactions beyond the primary specificity pocket of the enzyme. A paradigm widely accepted in the field is that macromolecular specificity in thrombin and related clotting proteases is achieved and controlled by interaction with "exosites," i.e. domains widely separated from the active site region that kinetically control docking of substrate in ways that restrict choices by the enzyme (8, 9). However, abundant mutagenesis data show that residues within the active site play roles that far exceed in importance those of exosites and, in fact, affect specificity and the choice of which substrate can be cleaved by the enzyme in ways that are unmatched by other domains (10 -13). Consistent with these findings, the present study demonstrates that residue 215 within the thrombin active site is th...

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Section: Methodssupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Engineering Thrombin for Selective Specificity toward Protein C and PAR1

Marino

Pelc

Vogt

et al. 2010

Journal of Biological Chemistry

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“…The E*-E equilibrium also provides context to interpret the effect of mutations associated with loss of biological activity in highly active proteases. In some cases, as documented by thrombin (8,58), the molecular origin of the effect is unclear because the mutation does not affect residues in direct contact with substrate. Stabilization of E* through molecular conduits not necessarily involved in substrate recognition may offer a plausible explanation.…”

Section: Discussionmentioning

confidence: 99%

Stabilization of the E* Form Turns Thrombin into an Anticoagulant

Bah

Carrell

Chen

et al. 2009

Journal of Biological Chemistry

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Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 Å resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 Å ) to the recently identified E* form. The side chain of Trp 215 collapses into the active site by shifting >10 Å from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu 192 -Gly 193 peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.

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“…Alternatively, elimination of Glu149 might alter domain-domain intramolecular interactions and promote conformational changes in the protease domain, thereby facilitating APC's interactions with fVa, similar to the effects achieved by protein S. 49 Clues toward the structural implications of the Glu149 to Ala mutation might be derived from the crystal structure of thrombin and from Ala scanning mutagenesis of the thrombin A-chain. 50,51 Glu8 in thrombin (chymotrypsin numbering), homologous to Glu149 in the C-terminal end of the protein C light chain, contributes to an ion quartet implicated in stabilizing the position of A-chain residues to form numerous interactions with the thrombin B-chain. An ionic interaction of Glu8 with Lys202, which is only 7 residues away from the active site Ser195, provides a rationale how Ala substitution of Glu8 in thrombin modulates specificity of the active site more than 20 Å away.…”

Section: Discussionmentioning

confidence: 99%