Tryptophans 231 and 234 in protein C report the Ca(2+)-dependent conformational change required for activation by the thrombin-thrombomodulin complex.
Human protein C circulates as both single- and two-chain zymogens. Activation by the physiological activation complex, thrombin-thrombomodulin, generates the anticoagulant enzyme, activated protein C. Ca2+ binding to the protease domain of protein C is accompanied by 5.5 +/- 0.2% quenching of intrinsic fluorescence that correlates with the conformational change required for the rapid activation by the thrombin-thrombomodulin complex. To map which Trp residues report this Ca2+ binding, candidate Trp residues at positions 84, 115, 145, 205, 231, and 234 were changed individually to Phe within a protein C deletion mutant lacking the Gla domain (GDPC). Of these, the Trp to Phe mutation at position 231 (W231F) eliminated the Ca(2+)-induced fluorescence quenching, and the Trp 234 to Phe mutation (W234F) increased the maximum quenching in protein C to 9.4 +/- 0.4%. Upon Ca2+ binding, the fluorescence emission intensity of the W231F mutant was increased 3.4% +/- 0.6%. The Kd for this site (84 +/- 20 microM) was similar to that of GDPC (Kd = 39 +/- 4 microM). To compare the properties of single- and two-chain protein C, we replaced the Lys156-Arg157 dipeptide cleavage site in protein C with Thr and Gln to form GDPCKR/TQ. GDPCKR/TQ and the two-chain form of protein C were activated at the same rate with the thrombin-thrombomodulin complex, they exhibited similar Ca2+ dependence for both activation and fluorescence quenching, and these enzymes had the same chromogenic activity. In contrast to the zymogen form, activated human Gla-domainless protein C did not undergo a Ca(2+)-induced fluorescence change. These results indicate that the environment of Trp 231 and 234 within the Ca2+ binding loop of the protein C zymogen are perturbed by Ca2+ binding to the zymogen.
Rezaie
A R
AR
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Esmon
C T
CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics
D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D014364:Tryptophan; D013917:Thrombin; D002118:Calcium