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.

中文翻译：

---

对人类颗粒酶H的底物特异性的结构见解：新型RKR基序的功能作用。

人粒酶H（GzmH）在人NK细胞中组成性表达，在对肿瘤和病毒的先天免疫应答中起重要作用。GzmH是一种胰凝乳蛋白酶样丝氨酸蛋白酶。人们对其底物偏好及其底物识别机理了解甚少。为了提供对GzmH底物识别机制的结构见解，我们解析了D102N-GzmH突变体的单独晶体结构，并与十肽底物和分别抑制2.2Å，2.4Å和2.7的抑制剂复合。GzmH的S1口袋中的Thr（189），Gly（216）和Gly（226）特异性三联体定义了其对P1位置庞大的芳香族残基（Tyr和Phe）的偏好。值得注意的是，我们发现一个仅在GzmH中保守的不寻常的RKR基序（Arg（39）-Lys（40）-Arg（41））有助于定义S3'和S4'结合区，表明在P3'和P4'位点偏爱酸性残基。底物中的RKR基序或酸性P3'和P4'残基的破坏消除了GzmH的蛋白水解活性。我们设计了一种四肽氯甲基酮抑制剂Ac-PTSY-氯甲基酮，可以选择性和有效地阻断GzmH的酶促和细胞毒活性，为进一步研究GzmH的功能提供了有用的工具。