Abstract
We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 x 10(8) m(-1) min(-1) for HCII-heparin when compared with 2.36 x 10(8) m(-1) min(-1) with wild-type thrombin and 0.03-0.53 x 10(8) m(-1) min(-1) for HCII-dermatan sulfate when compared with 5.23 x 10(8) m(-1) min(-1) with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a "double bridge" mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the "hirudin-like" domain of HCII with thrombin exosite-1.
Highlights
We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans
Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02– 0.38 ؋ 108 M؊1 min؊1 for HCII-heparin when compared with 2.36 ؋ 108 M؊1 min؊1 with wild-type thrombin and 0.03– 0.53 ؋ 108 M؊1 min؊1 for HCII-dermatan sulfate when compared with 5.23 ؋ 108 M؊1 min؊1 with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans
Inhibition of Thrombin Mutants by HCII in the Absence of Glycosaminoglycans—We used an Ala-scanning site-directed mutagenesis approach to map the residues on thrombin essential for HCII inhibition
Summary
Thrombin interaction [15,16,17, 28, 30]. Thrombin exosite-1 binds to the first acidic region of HCII to facilitate proteolytic attack of the P1-P1Ј Leu-Ser of HCII by the active site of thrombin. Thrombin is covalently trapped in a 1:1 stable bimolecular complex with HCII [23]. This information further illustrates the importance of both the acidic domain of HCII and the exosite-1 of thrombin, the molecular details and the role of individual surface-derived residues of thrombin are incomplete. We used 55 recombinant thrombin mutants in which solvent-accessible residues were substituted with Ala to determine the residues important for the thrombin-HCII interaction in the absence and presence of glycosaminoglycans. Our data support a mechanism for thrombin inhibition in the presence of glycosaminoglycan that involves binding of both proteins to the glycosaminoglycans and relies on the interaction of the amino-terminal acidic domain of HCII with exosite-1 for maximal rates of inhibition
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