Structure-function assessment of the role of the helical stack domain in the properties of human recombinant protein C and activated protein C.
The role of the helical stack (HS) in defining the properties of human recombinant (r) protein C (PC) and activated protein C (APC) was assessed. To do so, several mutations were made in this region of the molecule and their effects on the proteins examined. Substitution of the entire HS of PC (residues 38-46) by that of human coagulation factor (f) IX (residues 39-47), yielding r-[HSIX]PC, did not result in any substantial changes in the gamma-carboxyglutamic acid domain (GD)-related Ca(2+)-dependent properties of PC or APC, suggesting that the conformation of the HS may play a more dominant role in these Ca(2+)-dependent properties than do the specific amino acids that differ between these two HS regions. On the other hand, the catalytic efficiency of activation of r-[HSIX]PC by the thrombin/thrombomodulin complex was reduced to approximately one-third of that of wtr-PC, a result that demonstrates a specific role for the HS of PC in this activation process. Another mutation, [Ser42-->Pro], was generated in the HS region of r-PC, providing r-[S42P]PC, a change that according to the empirical algorithm based on the Chou-Fasman secondary structure rules, would disrupt the alpha-helical conformation of the HS. The anticoagulant activity of the corresponding r-[S42P]APC was found to be approximately 35% of that of wtr-APC. Because of the lack of any notable effects of this mutation on other GD-related Ca(2+)-dependent properties of r-PC and r-APC, the basis of this anticoagulant activity loss may be due to its nonmaximal alignment with substrate on the PL surface. The results of this study indicate that the role of the HS of r-PC and r-APC is to provide a region of the protein that is needed to assure optimal alignment on the PL or cell surface of the active site of the enzyme with that of the cleavage sites of the substrates, perhaps by functioning as a scaffold for separation of the active site of APC from the PL surface.
Christiansen
W T
WT
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Geng
J P
JP
Castellino
F J
FJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D011486:Protein C; D011994:Recombinant Proteins; D002118:Calcium