Structure-function and refolding studies of the thrombin-specific inhibitor hirudin.
We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.
Johnson
P H
PH
Molecular Biology Department, SRI International, Menlo Park, Calif.
Sze
P
P
Winant
R C
RC
Hudson
D
D
Underhill
P
P
Lazar
J B
JB
Olsen
C
C
Almquist
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002850:Chromatography, Gel; D005340:Fibrinogen / Q000378:metabolism; D006629:Hirudins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism
D006629:Hirudins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005340:Fibrinogen; D013917:Thrombin; C060225:desirudin