Structural Insights into the Substrate Specificity of Human Granzyme H: The Functional Roles of a Novel RKR Motif
Structural Insights into the Substrate Specificity of Human Granzyme H: The Functional Roles of a Novel RKR Motif
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DOI：
10.4049/jimmunol.1101381
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发表时间：
2012-01
期刊：
影响因子：
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通讯作者：
Li Wang;Kai Zhang;Lianfeng Wu;Shengwu Liu;Honglian Zhang;Qiangjun Zhou;L. Tong;F. Sun;Z. Fan
中科院分区：
文献类型：
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作者：
Li Wang;Kai Zhang;Lianfeng Wu;Shengwu Liu;Honglian Zhang;Qiangjun Zhou;L. Tong;F. Sun;Z. Fan
人颗粒酶H （GzmH）在人NK细胞中组成性表达，在对抗肿瘤和病毒的先天免疫应答中起重要作用。GzmH是一种凝乳胰蛋白酶样丝氨酸蛋白酶。其底物偏好和底物识别机制尚不清楚。为了对GzmH的底物识别机制提供结构上的见解，我们分别解决了D102N-GzmH突变体单独和与十肽底物和抑制剂复合物的晶体结构，分别达到2.2 Å， 2.4 Å和2.7 Å。GzmH S1口袋中的Thr189、Gly216和Gly226特异性三联体决定了它对P1位置上的大体积芳香残基（Tyr和Phe）的偏好。值得注意的是，我们发现了一个不寻常的RKR基序（Arg39-Lys40-Arg41），仅在GzmH中保守，有助于定义S3 ‘和S4 ’结合区，表明P3 ‘和P4 ’位点偏爱酸性残基。RKR基序或底物中酸性P3 ‘和P4 ’残基的破坏使GzmH的蛋白水解活性消失。我们设计了一种四肽氯甲基酮抑制剂ac - ptsy -氯甲基酮，可以选择性有效地阻断GzmH的酶活性和细胞毒活性，为进一步研究GzmH的功能提供了有用的工具。
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 Thr189, Gly216, and Gly226 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 (Arg39-Lys40-Arg41), 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.