Ratia Kiira, Kilianski Andrew, Baez-Santos Yahira M, Baker Susan C, Mesecar Andrew
Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois, United States of America.
Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, United States of America.
PLoS Pathog. 2014 May 22;10(5):e1004113. doi: 10.1371/journal.ppat.1004113. eCollection 2014 May.
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.
严重急性呼吸综合征冠状病毒（SARS-CoV）编码一种木瓜蛋白酶样蛋白酶（PLpro），它具有去泛素化（DUB）和去ISGylation活性，旨在抵消激活先天免疫反应的信号分子的翻译后修饰。在这里，我们研究了PLpro对泛素链和干扰素刺激基因15（ISG15）特异性的结构基础。我们展示了PLpro与泛素醛复合物的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。我们表明，PLpro对K48连接的泛素链的偏好远高于K63连接的泛素链，并且对基于ISG15的底物的偏好高于单泛素化底物。我们提出，PLpro对K48连接的泛素链和ISG15的更高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的手掌结构域中，最远端的泛素结构域与拇指结构域的“脊”区域相互作用。对该脊区域内残基的诱变表明，这些突变体保留了病毒蛋白酶活性以及催化单泛素水解的能力。然而，这些突变体中有一部分水解底物ISG15-AMC的能力显著降低，或者被K48连接的双泛素抑制。对于后一类残基，我们发现PLpro对活化B细胞的核因子κ轻链增强子（NFκB）信号通路的拮抗作用被消除。对PLpro中识别和处理双泛素和ISG15与单泛素和蛋白酶活性所需的关键和独特位点的鉴定，为泛素链和ISG15识别提供了新的见解，并突出了PLpro DUB和去ISGylase活性在先天免疫反应拮抗中的作用。
