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J. Biol. Chem., Vol. 279, Issue 37, 38519-38524, September 10, 2004

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The Conformation of the Activation Peptide of Protein C Is Influenced by Ca²⁺ and Na⁺ Binding^*

Likui Yang, Swati Prasad, Enrico Di Cera, and Alireza R. Rezaie¶

From the the Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104 and Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri 63110

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca²⁺. To provide direct evidence for the linkage between Ca²⁺ binding and the conformation of the activation peptide, we have constructed a protein C mutant in the -carboxyglutamic acid-domainless form in which the P1 Arg¹⁶⁹ of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca²⁺, the intrinsic fluorescence of the mutant decreases 30%, as opposed to only 5% for the wild-type, indicating that Trp¹⁶⁹ is directly influenced by the divalent cation. The K_d of Ca²⁺ binding for the mutant protein C was impaired 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 5-fold in the presence of Ca²⁺. These findings suggest that Ca²⁺ 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.

Received for publication, June 30, 2004 , and in revised form, July 14, 2004.

^* This work was supported by National Institutes of Health Grants HL68571 and HL62565 (to A. R. R.) and HL49413, HL58141, and HL73813 (to E. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-977-9240; Fax: 314-977-9205; E-mail: rezaiear{at}slu.edu.

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