br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion CHIKV is an important arthropod borne virus with substantial impact on global health and cause a major viral disease that requires the development of antiviral drug to combat chikungunya disease. Virus specific proteases have particularly become an attractive drug targets for viral diseases, ever since the potent protease inhibitor specific to HIV-1 protease have been licensed and protease inhibitor for hepatitis C virus have entered the clinical trials [54,55]. The C-terminal domain of nsP2 (nsP2pro) is a papain-like cysteine protease that plays a key role in processing of the viral non-structural polyprotein inside the infected host cell and leads to the formation of individual virus replication proteins (nsP1, nsP2, nsP3 and nsP4) [25,52]. Till date, no drug is commercially available for the treatment of chikungunya infection. CHIKV specific viral enzymes, including two of the proteases the trpv1 antagonist protease and the nsP2pro, are potential target for antiviral drug development and discovery [47,56]. In this study we have described the detailed crystal structure analysis of CHIKV nsP2pro. Our investigation gained insights into conformational variability of the active site and the understanding of molecular determinants of CHIKV nsP2pro enzymatic activity and substrate specificity. Crystal structure of CHIKV nsP2pro is observed to have four molecules in the asymmetric unit and each subunit consists of two subdomains; the N-terminal protease subdomain and the C-terminal MTase-like subdomain. The catalytic pocket of the enzyme is positioned at interface of the protease and the MTase-like subdomains. The surface view of the crystal structure of CHIKV nsP2pro revealed that the active site at the interface is closed because of high flexibility of the loop present between β1 and β2 strands of the protease subdomains. This loop between β1 and β2 strands contains Asn547 residue that has high conformational variability and elasticity, due to which Asn547 seems to be regulating the access of the active site by specifically binding to the substrate subsite. Previous molecular docking and simulation studies of VEEV nsP2pro with the substrate peptides showed that this β-hairpin might restricts the entry of the substrate into the active site. This observation was based on the highly dynamic nature of the β-hairpin between β1 and β2 strands and the interaction of the β-hairpin residue, Asn545 with the docked peptide in VEEV nsP2pro [27]. Structural comparison analysis of the active site cleft of CHIKV nsP2pro with that of VEEV nsP2pro revealed the substrate binding position and has led to the identification of the active site groove residues that participate in the substrate binding (Table 2). The prefixes P and S refer to the substrate peptide residues and the complementary protease binding sites respectively [41]. In CHIKV nsP2pro structure these sites appear as shallow depression in a deep long groove at the interface of the protease and the MTase-like subdomains (Fig. 5A, B). There are number of residues in the substrate binding cleft along with the catalytic His548 and Cys478 that contribute to each of these subsites (Table 2). It has been observed that most of the residues are conserved in these subsites in the alphaviruses (Fig. S3). The residues of S1′ and S1 subsites of CHIKV nsP2pro are exactly same as in VEEV nsP2pro protease while few differences are observed in the residues of the S2, S3 and S4 subsites in two structures [27,28]. Three dimensional structural comparison of CHIKV nsP2pro with VEEV nsP2pro reveals that the P1 residue of substrate binds to the S1 subsite of nsP2pro, which is formed by catalytic Cys478 and nearby residues Asn476, Val477and Trp479. The S2 subsite is defined by Trp549, a highly conserved residue in alphaviruses along with Asn547, Trp479 and Tyr512. The conservation of Trp549 in alphavirus nsP2pro and its interaction with Gly at P2 position makes CHIKV nsP2pro a GSM specific cysteine protease [57]. The P3 Ala of CHIKV substrate peptide binds to S3 subsite residue of nsP2pro which are Asn547, Tyr544 and Met707, while the S4 subsite is defined by Trp549, Gln706 and Asp711. Structural analysis revealed that the access to the active site and substrate binding groove is blocked by a dynamic loop present in the protease subdomain between the β1 and β2 strands. This loop contains a substrate binding residue Asn547, side chain of which is flexible and open to attain more than one conformation, as evident by its high B factor. Previously concluded molecular docking and biochemical studies have revealed the significance of Asn547 as a substrate binding residue in VEEV nsP2pro and CHIKV nsP2pro. In the crystal structure of CHIKV nsP2pro this residue is found coming towards Leu670 of the MTase-like domain and closing the access of active site. To examine the role of this residue in substrate recognition and binding Asn547 was mutated to Ala which results in a three-fold drop in Kcat/Km. This reduction in catalytic efficiency may account for the specificity of Asn547 as a substrate binding residue, which is involved not only in main chain interactions with the substrate but it's side chain might also participate in binding interaction to the substrate subsites. These observations reflect that conformational flexibility of the loop connecting β1 and β2 strands might be responsible for regulating the access to the active site residue Cys478, which lies just beneath this variable loop in the crystal structure of CHIKV nsP2pro.