title: Structural insights into the substrate specificity of human granzyme H: the functional roles of a novel RKR motif.
abstract: Human granzyme H (GzmH) is constitutively expressed in human NK cells that have important roles in innate immune responses against tumors and viruses. GzmH is a chymotrypsin-like serine protease. Its substrate preference and its mechanism of substrate recognition are poorly understood. To provide structural insights into the substrate recognition mechanisms for GzmH, we solved the crystal structures of a D102N-GzmH mutant alone and in complex with a decapeptide substrate and an inhibitor to 2.2 Å, 2.4 Å, and 2.7 Å, respectively. The Thr(189), Gly(216), and Gly(226) specificity triad in the S1 pocket of GzmH defines its preference for bulky, aromatic residues (Tyr and Phe) at the P1 position. Notably, we discovered that an unusual RKR motif (Arg(39)-Lys(40)-Arg(41)), conserved only in GzmH, helps define the S3' and S4' binding regions, indicating the preference for acidic residues at the P3' and P4' sites. Disruption of the RKR motif or the acidic P3' and P4' residues in the substrate abolished the proteolytic activity of GzmH. We designed a tetrapeptide chloromethylketone inhibitor, Ac-PTSY-chloromethylketone, which can selectively and efficiently block the enzymatic and cytotoxic activity of GzmH, providing a useful tool for further studies on the function of GzmH.
authors.affiliation: Chinese Academy of Sciences Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.forename: Lianfeng
authors.initials: L
authors.lastname: Wu
authors.forename: Shengwu
authors.initials: S
authors.lastname: Liu
authors.forename: Honglian
authors.initials: H
authors.lastname: Zhang
authors.forename: Qiangjun
authors.initials: Q
authors.lastname: Zhou
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
authors.forename: Zusen
authors.initials: Z
authors.lastname: Fan
mesh_terms: D020816:Amino Acid Motifs / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000187:drug effects / Q000276:immunology; D002415:Cats; D002460:Cell Line; D002461:Cell Line, Transformed; D018360:Crystallography, X-Ray; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004285:Dogs; D053804:Granzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020014:K562 Cells; D008969:Molecular Sequence Data; D002679:Pan troglodytes; D011485:Protein Binding / Q000276:immunology; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D013379:Substrate Specificity / Q000276:immunology
chemical_list: D015842:Serine Proteinase Inhibitors; C069434:GZMH protein, human; D053804:Granzymes