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* Center for Infection and Immunity, and
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and
College of Life Sciences, Nankai University, Tianjin, China
Granzyme M (GzmM), a unique serine protease constitutively expressed in NK cells, is important for granule-mediated cytolysis and can induce rapid caspase-dependent apoptosis of tumor cells. However, few substrates of GzmM have been reported to date, and the mechanism by which this enzyme recognizes and hydrolyzes substrates is unknown. To provide structural insights into the proteolytic specificity of human GzmM (hGzmM), crystal structures of wild-type hGzmM, the inactive D86N-GzmM mutant with bound peptide substrate, and the complexes with a catalytic product and with a tetrapeptide chloromethylketone inhibitor were solved to 1.96 Å, 2.30 Å, 2.17 Å and 2.70 Å, respectively. Structure-based mutagenesis revealed that the N terminus and catalytic triad of hGzmM are most essential for proteolytic function. In particular, D86N-GzmM was found to be an ideal inactive enzyme for functional studies. Structural comparisons indicated a large conformational change of the L3 loop upon substrate binding, and suggest this loop mediates the substrate specificity of hGzmM. Based on the complex structure of GzmM with its catalytic product, a tetrapeptide chloromethylketone inhibitor was designed and found to specifically block the catalytic activity of hGzmM.
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.
1 This work was supported by the National Science Foundation of China (30525005, 30830030, 30623005, and 30772496), 863 program (2006AA02Z4C9, 2006AA02Z173), 973 programs (2006CB504303, 2006CB806506 and 2006CB910901), the Chinese Academy of Sciences (KSCX2-YW-R-42 and the Hundred Talents Program), and the support of K.C. Wong Education Foundation (Hong Kong) to Z.F.
2 These authors contributed equally to this work.
3 Address correspondence and reprint requests to: Dr. Fei Sun or Dr. Zuse Fan, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. E-mail address: feisun{at}ibp.ac.cn or fanz{at}moon.ibp.ac.cn
4 Abbreviations used in this paper: Gzm, granzyme; aM, active hGzmM; aM-Inhibitor, active GzmM bound to its inhibitor; aM-Prod, active GzmM in complex with its product; CMK, chloromethylketone; CPEP, cleaved peptide; 
II-Gzm, truncated hGzmM; G3P-GzmM, Gly3 mutated to Pro; hGzm, human Gzm; I:E, molar inhibitor to enzyme; OPEP, octa-peptide; P, substrate amino acid; PI, propidium iodide; S, substrate binding site.
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