MAPPING CONFORMATIONAL TRANSITIONS OF CHIKUNGUNYA NSP2 HELICASE THROUGH ENHANCED SAMPLING SIMULATIONS

Chikungunya, among others, is a virus that causes a debilitating fever with no specific treatment. Viral replication requires RNA unwinding by the nsP2 helicase. NsP2 is multifunctional: its N-terminal domain harbors helicase and NTPase activity, while the C-terminal domain has protease function. Helicase activity is abolished when nsP2 lacks the protease region, despite retained ATPase activity, suggesting an unclear mechanistic role for the protease in RNA unwinding. The catalytic core consists of two RecA-like domains whose interface binds NTP. NTP binding drives domain closure, and subsequent hydrolysis and product release reopen the cavity, repositioning the enzyme along the nucleic acid. Repeated cycles produce stepwise translocation, destabilizing base-pairing in double-stranded RNA. To map the free energy landscape of RecA-like domain transition, we employed String Method with Swarms of Trajectories (SMwST) coupled with extended-system adaptive biasing force (eABF). SMwST identifies a meaningful transition pathway in collective variable (CV) space by running multiple short unbiased simulations from discretized path images, from which a mean drift evolves the string toward the optimal pathway. The resulting string defines a pathway collective variable (PCV) used as the reaction coordinate for free energy calculations. In eABF, the average force along the PCV is estimated and canceled via a fictitious particle harmonically coupled to the system, enabling simultaneous barrier removal and PMF calculation. Simulations were run in NAMD 3 with ff19SB, OL3 and OPC. The free energy profiles along RecA-like domain closure are expected to reveal how ATP binding modulates the energetic barriers of the conformational transition. Comparison between full-length nsP2 and the isolated helicase domain should expose if there are differences in the free energy landscape attributable to the protease region, providing a quantitative basis for the observed loss of helicase activity. The obtained results will contribute to the mechanistic understanding of nsP2 helicase activity and the role of the protease domain, potentially informing the design of antiviral strategies targeting the Chikungunya replication machinery.