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The Human Apoptosis-Inducing Granzyme M Structural Basis For Structural Basis for Proteolytic Specificity of the Human Apoptosis-Inducing Granzyme M Lianfeng Wu, Li Wang, Guoqiang Hua, Kan Liu, Xuan Yang, Yujia Zhai, Mark Bartlam, Fei Sun and Zusen Fan This information is current as of September 29, 2021. J Immunol 2009; 183:421-429; ; doi: 10.4049/jimmunol.0803088 http://www.jimmunol.org/content/183/1/421 Downloaded from References This article cites 42 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/183/1/421.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Structural Basis for Proteolytic Specificity of the Human Apoptosis-Inducing Granzyme M1 Lianfeng Wu,*† Li Wang,*† Guoqiang Hua,*† Kan Liu,*† Xuan Yang,*† Yujia Zhai,† Mark Bartlam,‡ Fei Sun,2,3† and Zusen Fan2,3*† 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 Downloaded from 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 Journal of Immunology, 2009, 183: 421–429. 4 ranzyme (Gzm) -induced cell death is a major pathway plays very important roles in granule-mediated cytolysis and can http://www.jimmunol.org/ used by CTL and NK cells to eliminate virus-infected or induce rapid cell death via an as yet undefined mechanism (13, 14). G transformed tumor cells (1, 2). Gzms are normally ex- Several substrates have been identified for GzmM so far, including pressed in an inactive prostate called Pro-Gzm, and the N terminus the GzmB serpin PI9 (15), the inhibitor of caspase-activated of Pro-Gzm is subsequently cleaved to release the active form with DNase (14), the reactive oxygen species antagonist TRAP1 (16), the constitutive N-terminal sequence IIGG. Five types of human the component of cytoskeleton ␣-tubulin (17), and the abundant Gzms (A, B, H, K, and M) have been identified to date. GzmA and nucleolar phosphoprotein nucleophosmin (18). B are the most abundant Gzms in CTLs and lymphokine-activated Human Gzms share high sequence homology and similar struc- killer cells and have been extensively studied (3–7). tures. However, positional screening techniques determined that they GzmM, a chymotrypsin-like serine protease, preferentially cleaves possess distinctive substrate specificities (8). High-resolution struc- by guest on September 29, 2021 its substrate after Met or Leu (8, 9). Human Gzm (hGzm)M is en- tures of the Gzms are indispensable for further investigation of their coded in a distinctive cluster on chromosome 19 and colocalizes specific substrate binding sites. To date, the three-dimensional struc- with a family of neutrophil elastases (10). GzmM is constitutively tures of human GzmA, B, and Pro-GzmK are known and have re- and highly expressed in activated NK cells, but is never detected vealed the structural basis for their substrate recognition (19–22), yet in CD4ϩ or CD8ϩ T cells even after activation (11, 12). GzmM no structures are available for hGzmM and hGzmH. To elucidate the substrate-binding specificity and catalytic mech- anisms of GzmM, we have determined the crystal structures of wild- *Center for Infection and Immunity, and †National Laboratory of Biomacromol- type hGzmM, the inactive D86N-GzmM mutant bound with a peptide ecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and substrate (D86N-Sub), hGzmM in complex with a catalytic product ‡ College of Life Sciences, Nankai University, Tianjin, China (aM-Prod), and hGzmM in complex with a tetrapeptide CMK inhib- Received for publication September 17, 2008. Accepted for publication April itor (aM-Inhibitor) to 1.96 Å, 2.30 Å, 2.17 Å, and 2.70Å, respectively. 25, 2009. Based on our structural analysis, we generated a series of mutants to 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 characterize the determinants of hGzmM enzymatic activity and with 18 U.S.C. Section 1734 solely to indicate this fact. found that Asp86 and His41 in the catalytic triad contribute more to 1 This work was supported by the National Science Foundation of China (30525005, proteolytic activity than the attack residue Ser182. We also found the 30830030, 30623005, and 30772496), 863 program (2006AA02Z4C9, 2006AA02Z173), D86N-GzmM mutant is an ideal catalytically inactive (dead) enzyme 973 programs (2006CB504303, 2006CB806506 and 2006CB910901), the Chinese Acad- emy of Sciences (KSCX2-YW-R-42 and the Hundred Talents Program), and the support for functional studies. From structural comparisons, we observed a of K.C. Wong Education Foundation (Hong Kong) to Z.F. large conformational change of the L3 loop upon substrate binding 2 These authors contributed equally to this work. and found this loop could endow the substrate with recognition and 3 Address correspondence and reprint requests to: Dr. Fei Sun or Dr. Zuse Fan, In- specificity. Based on the complex structure with a catalytic product, stitute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, we designed a tetrapeptide CMK inhibitor and found it can specifi- China. E-mail address: [email protected] or [email protected] cally block the catalytic activity of hGzmM. 4 Abbreviations used in this paper: Gzm, granzyme; aM, active hGzmM; aM-Inhib- itor, 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 Materials and Methods to enzyme; OPEP, octa-peptide; P, substrate amino acid; PI, propidium iodide; S, Plasmid construction for hGzmM and its variants substrate binding site. The cDNA fragments of Pro-hGzmM, active hGzmM, and the truncated Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 hGzmM (⌬II-GzmM) were amplified from the full length cDNA of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803088 422 CRYSTAL STRUCTURE OF HUMAN GRANZYME M Table I. Data collection, phasing, and refinement statisticsa aM D86N-Subb aM-Prod aM-Inhibitor Data collection Space group P3121 P3121 P3121 P3121 Unit cell: a, b, c (Å), ␣, ␤, ␥ (°) 74.4, 74.4, 113.2, 74.5, 74.5, 112.7, 74.6, 74.6, 112.9, 74.7, 74.7, 113.6, 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 Molecule in ASU 1111 Resolution (Å)c 50-1.96 (2.01-1.96) 50-2.30 (2.36-2.30) 50-2.17 (2.25-2.17) 57-2.70 (2.77-2.70) Completeness (%)c 98.5 (92.1) 99.4 (99.4) 98.9 (98.2) 99.8 (99.7) c,d Rmerge (%) 6.5 (34) 5.1 (39.9) 4.9 (44.8) 9.4 (40.8) I/␴ϽIϾb 28.0 (2.1) 36.1 (3.4) 20.5 (2.0) 7.3 (1.9) Refinement statistics d Rwork/Rfree (%) 20.9/25.7 24.0/28.8 21.2/25.0 22.3/29.3 r.m.s.d. Bond length (Å) 0.023 0.019 0.017 0.016 Bond angle (°) 1.726 1.859 1.641 1.880 Ramachandran plot (%) Most favored (%) 86.5 84.1 85.1 80.8 Allowed (%) 13.0 15.9 14.4 18.7 Generous allowed (%) 0.5 0 0.5 0.5 Downloaded from a Corresponding parameters for the highest-resolution shell are shown in parentheses. b D86N-Sub, D86N-GzmM bound to substrate (OPEP). c ϭ⌺⌺ ͉ ϪϽ Ͼ͉ ⌺ ⌺ Ͻ Ͼ Ͻ Ͼ Rmerge h i Iih Ih / h i Ih , where Ih is the mean of the observations Iih of reflection h. d ϭ⌺࿣ ͉ Ϫ ͉ ࿣ ⌺͉ ͉ ϭ Rwork ( Fp(obs) Fp(calc) )/ Fp(obs) ; Rfree R factor for a selected subset (5%) of the reflections that was not included in prior refinement calculations. hGzmM (RZPD German Resource Center for Genome Research) by stan- ␭ ϭ 1.5418 Å) at 93 K.
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