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J. Biol. Chem., Vol. 279, Issue 29, 30751-30759, July 16, 2004

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Characterization of Structural Determinants of Granzyme B Reveals Potent Mediators of Extended Substrate Specificity^*

Sandra Waugh Ruggles, Robert J. Fletterick¶||, and Charles S. Craik¶||**

From the Graduate Group in Biophysics and the Departments of ^¶Pharmaceutical Chemistry and ^||Biochemistry and Biophysics, University of California, San Francisco, California 94143-2280

Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile⁹⁹, Tyr¹⁷⁴, Arg¹⁹², and Asn²¹⁸. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile⁹⁹ completely defines and predicts P2 specificity, primarily through the binding constant K_m. Asn²¹⁸ variants have minor effects alone but in combination with mutations at Arg¹⁹² and Ile⁹⁹ alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn²¹⁸ variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn²¹⁸ not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members.

Received for publication, January 28, 2004 , and in revised form, April 23, 2004.

^* This work was supported by National Institutes of Health Grants CA72006 (to C. S. C.) and DK39304 (to C. S. C. and R. J. F.) and a University of California, San Francisco Chancellor's Research Award (to S. W. R.). 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.

Present address: Catalyst Biosciences Inc., 225 Gateway Blvd., South San Francisco, CA 94080.

^** To whom correspondence should be addressed: Dept. of Pharmaceutical Chemistry, University of California, 600 16th St., Box 2280, San Francisco, CA 94143-2280. Tel.: 415-476-9890; Fax: 415-502-8298; E-mail: craik{at}cgl.ucsf.edu.

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