Each nsP plays a distinct defined and

Each nsP plays a distinct, defined and indispensable role in the replication of viral genome. nsP1 is a membrane associated protein which possesses S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) and guanylyltransferase (GTase) activities to catalyze the viral RNA capping reaction [[11], [12], [13], [14], [15]]. nsP1 is also believed to interact with nsP4, facilitate the initiation of minus-strand RNA synthesis and target the replication complex to the plasma membrane by attaching it to the endoplasmic reticulum [[16], [17], [18], [19], [20], [21]]. nsP2 is a multifunctional protein, which is composed of two domains. The N-terminal domain possesses RNA helicase, RNA dependent 5′triphosphatase and nucleoside triphosphatase activity [[22], [23], [24]]. The C-terminal half of nsP2 is a cysteine protease, which cleaves the nonstructural polyprotein precursor nsP1234 into different nsPs in a specific and sequential manner [[25], [26], [27], [28], [29], [30]]. Besides, a nuclear localization sequence is present in nsP2 that is responsible for partially transporting nsP2 into the nucleus of virus infected host WYE-132 [31]. nsP3 is composed of two domains, a highly conserved N-terminal macrodomain and a less conserved C-terminal domain [32,33]. Recent studies have shown that macrodomain of nsP3 possesses ADP-ribosylhydrolase activity which is pivotal for viral life cycle [34,35]. nsP4, the RNA dependent RNA polymerase is the key enzyme responsible for catalyzing the synthesis of the viral RNAs, and it also possesses terminal adenylyltransferase activity (TATase) activity [36,37]. Viral cysteine proteases are a class of protease enzymes, which cleave viral polyproteins at specific site through the catalytic mechanism that involves nucleophilic cysteine thiol in the catalytic dyad or triad. CHIKV nsP2pro, the alphavirus cysteine protease belongs to the peptidase family C9 of clan CA, cleaves and processes the nonstructural polyprotein into various nsPs [38]. Active site of the enzyme contains a catalytic dyad composed of a Cys and a His residue. CHIKV nsP2pro has 40% sequence identity with VEEV nsP2pro (PDB ID: 2HWK) and 44% sequence identity with SINV nsP2pro (PDB ID: 4GUA). Alphavirus nsP2pro structure revealed that it contains a novel fold that does not structurally resemble any of the cysteine proteases but the relative positioning of the catalytic Cys and His dyad in the active site resembles the position of catalytic residues in the papain-like cysteine proteases [28,39]. Thus, CHIKV nsP2pro being virus specific is a potential antiviral drug target. This study reports the bacterial expression, purification, enzyme activity, effect of Asn547 mutation on substrate binding and catalysis, crystallization and atomic structure determination of CHIKV nsP2pro (471–791 residue) at 2.59 Å. The enzymatic activity of purified nsP2pro protein was confirmed by using a FRET based proteolytic assay. Crystal structure reveals that it contains an N-terminal subdomain possessing catalytic residues and a C-terminal subdomain that structurally resembles the SAM-dependent MTase domain. Structural analysis revealed the presence of a variable loop barricading the substrate binding cleft present at the interface of the two subdomains. This variable loop contains Trp549 and Asn547, and substrate docking studies for VEEV nsP2pro indicate their role in substrate recognition [27]. Significance of these residues in active site accessibility and modulation by possibly restricting the entry of substrate in the active site has previously been hypothesized by Russo et al., 2010 [27]. In this study, the crystal structure of CHIKV nsP2pro clearly reveals that the flexible loop containing Asn547 restricts the entry of substrate and substitution of Asn547 to Ala leads to reduction in enzymatic activity, specifying the importance of Asn547 in substrate recognition and binding. Further, the structural comparison of CHIKV nsP2pro with the crystal structures of VEEV and SINV nsP2pro illustrates the molecular specifics of the substrate binding cleft at the subdomain interface.