Mutation of Glu-80-->Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex.
Binding Ca2+ to a high affinity site in protein C and Gla-domainless protein C (protein C lacking residues 1-44) results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. Protein C modeling studies suggested the single high affinity Ca2+ binding-site might be present in a loop in the protease domain and involve Glu-70 and -80 (chymotrypsin numbering system). This loop, which is a known Ca(2+)-binding site in trypsin, is also conserved in other coagulation proteases, including factors VII, IX,and X. In thrombin, which does not bind Ca2+, Glu-70 is replaced by Lys, creating an internal salt bridge with Glu-80. We constructed and expressed a Gla-domainless protein C mutant in which Glu-80 is replaced with Lys. The activation of the resultant mutant is accelerated by thrombomodulin in a Ca(2+)-independent fashion. Unlike wild type Gla-domainless protein C, Ca2+ no longer inhibits activation of the mutant by free thrombin, and Ca2+ stimulation of chromogenic activity is also absent. The characteristic Ca(2+)-dependent quenching of Gla-domainless protein C intrinsic fluorescence is also absent in the mutant. We conclude that the high affinity Ca(2+)-binding site in protein C critical for zymogen activation involves Glu-80. The Glu-80 to Lys mutation probably results in a salt bridge with Glu-70 that stabilizes protein C zymogen in a conformation similar, if not identical, to the Ca(2+)-stabilized conformation favorable for rapid activation by the thrombin-thrombomodulin complex.
Rezaie
A R
AR
Oklahoma Medical Research Foundation, Oklahoma City 73104.
Mather
T
T
Sussman
F
F
Esmon
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D017931:DNA Primers; D004789:Enzyme Activation; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D005971:Glutamates; D018180:Thrombomodulin; D018698:Glutamic Acid; D014357:Trypsin; D013917:Thrombin; D008239:Lysine; D002118:Calcium