Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

Abstract RNA helicases encoded by positive-strand RNA viruses are essential for genome replication, yet the specific biological functions and mechanochemical basis underlying these functions remain poorly defined. Progress has been limited by the difficulty of resolving individual catalytic steps under single-turnover conditions, which are often experimentally inaccessible for viral enzymes. Alphaviruses replicate within membrane-bound spherules that may alter local metabolite concentrations, raising the possibility that the enzymatic properties of alphaviral proteins differ from those of viruses with greater cytosolic exposure. Here, we present a kinetic and binding analysis of full-length non-structural protein 2 (nsP2) from Chikungunya virus, a multifunctional superfamily 1B NTPase and RNA helicase. Purified nsP2 binds nucleoside triphosphates with high affinity, exhibiting equilibrium dissociation constants in the single digit micromolar range. This property enabled single-turnover, pre-steady-state, and isotope-trapping experiments that are rarely feasible for viral helicases. These analyses identified two sequential conformational-change steps required for nucleotide hydrolysis. Molecular dynamics simulations suggest tightening of the RecA1 and RecA2 domains upon ATP binding followed by compaction of the enzyme mediated by interactions between the 1B subdomain and RecA2 domain. Product inhibition patterns support random release of ADP and inorganic phosphate, with relative binding affinities indicating that ADP dissociates first. The reaction is irreversible. Although nsP2 binds RNA tightly, strand separation under single-turnover conditions is too slow to represent ATP-driven unwinding, instead likely reflecting formation of an unwinding-competent nsP2-RNA complex. Together, these findings establish a quantitative framework for nsP2 function and provide a roadmap for mechanistic studies of alphaviral helicases. Competing Interest Statement The authors have declared no competing interest. Data Availability All data are incorporated into the article and its online supplementary material. Constructs and data sets presented in this study are available upon request. Abbreviations - aMD - accelerated molecular dynamics - ATP - adenosine triphosphate - ATPγS - adenosine 5′-O-(3-thiotriphosphate) - CHIKV - Chikungunya virus - cMD - classical molecular dynamics - CTP - cytidine triphosphate - dsRNA - double-stranded RNA - EDTA - ethylenediaminetetraacetic acid - FRET - Forster resonance energy transfer - GTP - guanosine triphosphate - HCV - hepatitis C virus - HDXMS - hydrogen deuterium exchange mass spectrometry - HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - KCl - potassium chloride - MD - molecular dynamics - NMR - nuclear magnetic resonance - NPT - constant pressure and temperature ensemble - NTP - nucleoside triphosphate - NTPase - nucleoside triphosphate hydrolase - NVT - constant volume and temperature ensemble - PAGE - polyacrylamide gel electrophoresis - PCA - principal component analysis - PEI - polyethylenimine - Pi - inorganic phosphate - PMEMD - Particle Mesh Ewald Molecular Dynamics - RMSF - root mean square fluctuation - RNA - ribonucleic acid - RTPase - RNA triphosphatase - SF1B - superfamily 1B - SUMO - small ubiquitin-like modifier - TBE - Tris-borate-EDTA - TCEP - tris(2-carboxyethyl)phosphine - TLC - thin-layer chromatography - TPP - tripolyphosphate - UTP - uridine triphosphate - VMD - Visual Molecular Dynamics - WNV - West Nile virus - WT - wild type - ZV - Zika virus