Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like protease.
Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like protease.
复制标题
DOI：
10.1371/journal.ppat.1004113
复制
发表时间：
2014-05
期刊：
影响因子：
6.7
通讯作者：
Mesecar A
中科院分区：
文献类型：
--
作者：
Ratia K;Kilianski A;Baez-Santos YM;Baker SC;Mesecar A
严重急性呼吸综合征冠状病毒(SARS-CoV)编码一种木瓜蛋白酶(PLPro)，具有去泛素化(DUB)和去ISGyl化活性，被认为是为了对抗激活先天性免疫反应的信号分子的翻译后修饰。在这里，我们检查了PLPro的泛素链和干扰素刺激基因15(ISG15)特异性的结构基础。我们给出了PLPro与泛素醛形成的络合物的X射线晶体结构，并模拟了PLPro与其他泛素链和ISG15底物的相互作用。我们发现PLPro非常喜欢K48-连接的泛素链和ISG15基底物，而不是那些单一泛素化的底物。我们认为PLPro对K48连接的泛素链和ISG15具有较高的亲和力源于一种二价结合机制，其中两个泛素样结构域倾向于结合在PLPro的手掌结构域上，而最远端的泛素结构域与拇指结构域的“脊”区域相互作用。对该脊状区域内残基的突变表明，这些突变体保持了病毒蛋白水解酶的活性和催化单泛素的能力。然而，这些突变体中的一些显著降低了对底物ISG15-AMC的水解能力，或者被K48连接的二丁基奎宁抑制。对于这些残基，我们发现活化B细胞核因子-轻链增强剂(NF-κ-B)信号通路的PLPro拮抗作用被取消。PLPro中识别和处理二泛素和ISG15与单泛素和蛋白酶活性所需的关键和唯一位点的鉴定为了解泛素链和ISG15识别提供了新的见解，并强调了PLPro Dub和DeISGylase活性在拮抗先天性免疫反应中的作用。所有冠状病毒，如SARS病毒和最近发现的中东呼吸综合征(MERS)病毒，在其基因组中至少编码一种木瓜蛋白酶(PLPro)酶，该酶在病毒致病过程中具有两种不同的功能。第一个功能是将病毒多蛋白处理成病毒复制所必需的单独蛋白质。第二个功能是从宿主细胞蛋白中去除泛素和ISG15蛋白，这可能有助于冠状病毒短路宿主的先天免疫反应。测定了SARS病毒PLPro与人泛素类似物的三维结构，揭示了冠状病毒PLPro酶是如何在分子水平上将泛素和ISG15从宿主细胞蛋白中剥离出来的。突变PLPro和泛素相互作用中的一系列氨基酸残基，以确定哪些相互作用只对PLPro识别泛素和ISG15修饰的蛋白质重要，而对病毒多蛋白的识别和切割不重要。SARS PLPro与泛素-醛的3D结构为PLPro如何与泛素类分子相互作用提供了重要的新线索，并为对MERS等其他致命冠状病毒进行类似研究提供了分子路线图。
Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response. All coronaviruses such as the SARS virus and the recently identified Middle East Respiratory Syndrome (MERS) virus encode in their genomes at least one papain-like protease (PLpro) enzyme that has two distinct functions in viral pathogenesis. The first function is to process the viral polyprotein into individual proteins that are essential for viral replication. The second function is to remove ubiquitin and ISG15 proteins from host cell proteins, which likely helps coronaviruses short circuit the host's innate immune response. The 3-dimensional structure of SARS virus PLpro in complex with a human ubiquitin analog was determined and reveals how coronavirus PLpro enzymes strip ubiquitin and ISG15 from host cell proteins at the molecular level. A series of amino acid residues involved in interactions between PLpro and ubiquitin were mutated to identify which interactions are important only for the recognition of ubiquitin and ISG15 modified proteins by PLpro and not for recognition and cleaving of the viral polyprotein. The 3D structure of SARS PLpro with ubiquitin-aldehyde sheds significant new light into how PLpro interacts with ubiquitin-like molecules and provides a molecular road map for performing similar studies on other deadly coronaviruses such as MERS.
登录
查看更多内容
影响因子：
5.4
作者：
Harcourt, BH;Jukneliene, D;Baker, SC
通讯作者：
Baker, SC
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
Beal, RE;Toscano-Cantaffa, D;Pickart, CM
通讯作者：
Pickart, CM
冠状病毒类木瓜蛋白酶的去泛素化和干扰素拮抗活性
影响因子：
5.4
作者：
Clementz, Mark A.;Chen, Zhongbin;Baker, Susan C.
通讯作者：
Baker, Susan C.
DOI：
10.1073/pnas.1013388108
发表时间：
2011-02-08
影响因子：
11.1
作者：
James, Terrence W.;Frias-Staheli, Natalia;Mark, Brian L.
通讯作者：
Mark, Brian L.