Date Published: May 22, 2014
Publisher: Public Library of Science
Author(s): Kiira Ratia, Andrew Kilianski, Yahira M. Baez-Santos, Susan C. Baker, Andrew Mesecar, Félix A. Rey.
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.
Ubiquitin (Ub), a 76-amino-acid protein, is the building block for a set of versatile, post-translational modifications that regulate a number of cellular pathways, including many processes associated with combating viral infection . Through the action of activating and conjugating enzymes, the C-terminus of ubiquitin is covalently attached to the ε-amino group of lysine side chains on target proteins, forming an isopeptide bond. The most common ubiquitin modifications are extended to form chains of ubiquitin molecules, linked through a lysine side chain on the proximal ubiquitin and the C-terminus of the neighboring distal ubiquitin. The complexity associated with ubiquitin modifications arises from the ubiquitin lysine residue participating in the polyubiquitin chain. There are 7 lysine residues on ubiquitin, and most are believed capable of forming homotypic chains that mediate different linkage-dependent cellular pathways . The most prevalent ubiquitin linkage and most widely studied, the K48-based chain, directs the modified protein to the proteasome for degradation . Another well characterized Ub modification, the K63-based chain, is commonly associated with regulating endocytic processes, the DNA-damage response, and innate immune response pathways . More recently, the recognition of linear ubiquitin chains has been implicated in the activation of NFκB signaling .
Distinguishing the roles of multifunctional enzymes in viral replication is challenging. A critical first step is the proof-of-principle that an enzyme has distinct binding sites for different substrates. For SARS-CoV PLpro, an enzyme with peptidase and isopeptidase activities, the X-ray structure of PLpro in complex with ubiquitin aldehyde, and subsequent modeling of ubiquitin-chain and ISG15 interactions, suggested that a ridge region in PLpro is likely involved in binding ubiquitin-like modifier substrates. Analysis of PLpro mutants in protease and isopeptidase (DUB and DeISGylase) activity and competitive binding assays identified a hydrophobic portion of this ridge region in the SUb2 subsite that is essential for robust deISGylaing activity and interactions with ubiquitin chains, but is not required for protease or Ub-AMC hydrolysis activity. Thus, we were able to successfully decouple the viral protease processing activity of PLpro from its ubiquitin-chain and ISG15 hydrolysis activities.