The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding.
Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Prasad
Swati
S
Di Cera
Enrico
E
Rezaie
Alireza R
AR
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002412:Cations; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007477:Ions; D007700:Kinetics; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012964:Sodium / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000235:genetics
D002412:Cations; D007477:Ions; D010455:Peptides; D011486:Protein C; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D012964:Sodium; D002918:Chymotrypsin; D013917:Thrombin; D002118:Calcium