Protein Science zyxwvutsrqpon (1994). 3:588-599. Cambridge University Press. Printed in the USA. Copyright zyxwvutsrqp 0 1994 The Protein Society Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen CINDY L. FISHER, JUDITH S. GREENGARD, zyxwvu AND JOHN H. GRIFFIN Departments of Molecular and Experimental Medicine and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 (RECEIVED November 12, 1993; ACCEPTED February 9, 1994) Abstract Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying func- tional features relevant to the expression of their activities and specificities. The human serine protease anticoag- ulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phe- notypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models correspond- ing to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models in- cluded a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspec- tion of the surface features of the models allowedidentification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site re- gion was modeled and analyzed. Keywords: blood coagulation; calcium binding site; homology model; protein C; serine protease domain The human plasma factor protein C provides an important feed- back regulator of thrombosis because deficiencies of protein C can result in severe recurrent venous thrombotic disease (Griffin et al., 1981) treatable with protein C concentrates (Dreyfus et al., 1991). At least 2 clinical phenotypes are recognized. The most common form is autosomal dominant with variable penetrance (reviewed by Greengard and Griffin, 1988). Symptomatic pa- tients with this form of hereditary protein C deficiency most of- ten present with venous thrombosis in young adulthood; some family members, however, are symptom-free lifelong. Labora- tory workup of symptomatic heterozygous deficient patients re- veals -50% level of activity of plasmaprotein C, either accompanied by a proportionate reduction in the antigen level (Type I deficiency) or by the presence of antigen levels that sig- nificantly exceed activity levels, which implies the presence of circulating abnormal protein C molecules (Type zyxwvut I1 deficiency). A recessive form of protein C deficiency has been described in zyxwvut ~ " ~ _ _ ~ ~~ SCR-5, 10666 North Torrey Pines Road, La Jolla, California 92037; Reprint requests to: John H. Griffin, TheScripps Research Institute, e-mail: greengar@scripps.edu. homozygous or doublyheterozygous deficient infants who present with purpura fulminans or venous thrombosis shortly after birth (Branson et al., 1983; Seligsohn et al., 1984). The he- terozygous deficient relatives of these infants are usually clini- cally unaffected. Genetic analyses of DNA mutations in patients with these 2 clinical phenotypes of protein C deficiency have failed to reveal a genetic or an implied structural basis for the differences in clinical presentation (Reitsma et al., 1993). The biochemical regulation and action of protein C, a trypsin- like serine protease zymogen (Gardiner & Griffin, 1985; Esmon, 1987), is complex. Activated protein C exerts its antithrombotic effect through proteolysis of specific peptide bonds in the co- agulation cofactors factors Va and VIIIa (Vehar & Davie, 1980; Walker, 1981; Marlar et al., 1982; Fulcher et al., 1984), usually with the help of its nonenzymatic protein cofactor, protein S (Walker, 1980). In doing so it requires calcium ions and phos- pholipid surfaces or membranes for optimal activity (Walker, 1981; Bakker et al., 1992). Activated protein C is also known to bind to platelets and endothelial cells inthe presence of pro- tein S (Suzuki et al., 1984; Harris & Esmon, 1985; Hackeng et al., 1993). Protein zyxwv C is synthesized as a single-chain zymo- 588