NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

Abstract

Dengue genome encodes a two component protease complex (NS2B-NS3pro) essential for the viral maturation/infectivity, thus representing a key drug target. Previously, due to its “complete insolubility”, the isolated NS3pro could not be experimentally studied and it remains elusive what structure it adopts without NS2B and why NS2B is indispensable. Here as facilitated by our previous discovery, the isolated NS3pro has been surprisingly deciphered by NMR to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (PRE). The disordered NS3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. Moreover, we have in vitro refolded the NS3pro in complex with either NS2B (48–100) or the full-length NS2B (1–130) anchored into the LMPC micelle, and the two complexes have similar activities but different dynamics. We also performed molecular dynamics (MD) simulations and the results revealed that NS2B shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. Remarkably, the NS2B cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the SARS 3CL protease. Indeed, a truncated NS2B (48–100;Δ77–84) with the flexible loop deleted is able to trap the NS2B-NS3pro complex in a highly dynamic and catalytically-impotent state. Taken together, our study implies potential strategies to perturb the NS2B-NS3pro interface for design of inhibitors for treating dengue infection.

Unique properties of Zika NS2B-NS3pro complexes as decoded by experiments and MD simulations

“…The refolded NS2B-NS3pro with His-tag (column 2 of Fig. We attempted to refold Zika NS3pro without NS2B and found that NS3pro completely precipitated during refolding, thus indicating that Zika NS3pro also absolutely requests its NS2B cofactor for the correct folding, as previously observed on other flavivirus NS3 proteases (12)(13)(14)(15)(16)(17)(18). 1C).…”

“…Cloning, expression and purification of linked and unlinked Zika NS2B-NS3pro-Based on the sequence alignment with NS2B and NS3pro of the Dengue serotype 2 we previously characterized (12), the corresponding Zika sequences were identified for the NS2B (Fig. 1A) and NS3pro (Fig.…”

“…1A); as well as isolated NS3 (14-185) (Fig. The expression level of the recombinant NS2B was extremely low, and consequently we needed to grow many liters of E. coli cells to obtain sufficient NS2B for refolding with NS3pro by the same protocol we previously utilized for the Dengue one (12). The expression level of the recombinant NS2B was extremely low, and consequently we needed to grow many liters of E. coli cells to obtain sufficient NS2B for refolding with NS3pro by the same protocol we previously utilized for the Dengue one (12).…”

“…The correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and a viral NS2B-NS3 protease (NS2B-NS3pro) (10,(12)(13)(14)(15)(16)(17)(18). As a consequence, the flaviviral NS2B-NS3pro has been well-established to be a key target for developing antiviral drugs (10,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In the present study, we successfully obtained two enzymatically active Zika author/funder.…”

Host-Virus Interaction of ZIKA Virus in Modulating Disease Pathogenesis

“…The NS3 localizes to the membranes by interacting with the membrane-localizing transmembrane viral protein NS2B and helps in viral polyprotein processing and viral replication (Falgout et al, 1991; Markoff et al, 1997; Yu et al, 2013). The trypsin-like serine protease activity has been mapped to His53, Asp77, and Ser138 (catalytic triad) (Chambers et al, 1990; Gupta et al, 2015). …”