The C-terminus domain of non-structural 3 (NS3) protein of the Flaviviridae viruses (e. g. HCV, dengue, West Nile, Zika) is a nucleotide triphosphatase (NTPase) -dependent superfamily 2 (SF2) helicase that unwinds double-stranded RNA while translocating along the nucleic polymer. Due to these functions, NS3 is an important target for antiviral development yet the biophysics of this enzyme are poorly understood. Microsecond-long molecular dynamic simulations of the dengue NS3 helicase domain are reported from which allosteric effects of RNA and NTPase substrates are observed. The presence of a bound single-stranded RNA catalytically enhances the phosphate hydrolysis reaction by affecting the dynamics and positioning of waters within the hydrolysis active site. Coupled with results from the simulations, electronic structure calculations of the reaction are used to quantify this enhancement to be a 150-fold increase, in qualitative agreement with the experimental enhancement factor of 10–100. Additionally, protein-RNA interactions exhibit NTPase substrate-induced allostery, where the presence of a nucleotide (e. g. ATP or ADP) structurally perturbs residues in direct contact with the phosphodiester backbone of the RNA. Residue-residue network analyses highlight pathways of short ranged interactions that connect the two active sites. These analyses identify motif V as a highly connected region of protein structure through which energy released from either active site is hypothesized to move, thereby inducing the observed allosteric effects. These results lay the foundation for the design of novel allosteric inhibitors of NS3.
Flaviviruses (family Flaviviridae) are small (∼11 kilobases) positive-sense, single-stranded RNA (ssRNA) viruses that include members such as dengue (serotypes 1-4), Zika, West Nile, yellow fever, and Japanese Encephalitis viruses. The dengue virus (DENV) is a public health threat that causes serious morbidity and mortality globally [1,2]. Infection with DENV can result in “break-bone” fever, an extraordinarily painful disease with symptoms ranging from a mild fever to a fatal hemorrhagic syndrome [3]. There are approximately 50 million serious infections and 20,000 deaths each year, and dengue infections are a leading cause of mortality in children in a number of Latin and Asian countries [1]. Dengue viruses have re-emerged in the United States, and a growing number of locally acquired infections in Florida, Texas, and Hawaii have been reported over the last decade. Despite a reinvigorated effort due to the recent Zika epidemic [4], there are currently no approved small molecule antivirals to treat Flavivirus-induced diseases. One of the primary antiviral targets in Flaviviridae is the nonstructural protein 3 (NS3), which plays a critical role in the viral replication cycle [5–15]. NS3 is a multifunctional protein found in all Flaviviridae, possessing an N-terminal serine protease domain responsible for proteolytically cleaving the viral polyprotein during translation [16] and a C-terminal helicase/nucleotide triphosphatase (NTPase) /RNA triphosphatase domain [17–22]. In a nucleotide triphosphate (NTP) hydrolysis-dependent mechanism, the NS3 helicase domain (NS3h) unwinds double-stranded RNA (dsRNA) while translocating along the nucleic polymer. These functions are required to resolve the dsRNA replication intermediate into fully-mature positive strand RNAs (see Ref. [23] for a recent review). Mutations in the NS3 helicase and NTPase active sites are seen to abrogate NS3 function as well as decrease viral survival [24–26], demonstrating the importance of these enzymatic functions to the flavivirus life cycle. Drugs identified to inhibit DENV NS3h suffer from specificity issues because they are either NTPase inhibitors [27] or RNA/DNA mimics such as ivermectin [13], suramin [14] or aurintricarboxylic acid [15]. Therefore, it is of interest to further elucidate the mechanism of DENV NS3h with molecular resolution to help identify new and specific target regions for antiviral therapeutics. The Flaviviridae NS3h have been classified as a superfamily 2 (SF2) helicase (NS3/NPH-II subfamily; a DEx/H helicase) where the NTPase cycle (Fig 1) provides the free energy needed to unwind dsRNA and translocate along the nucleic substrate in a 3′ to 5′ direction [28]. Structurally, NS3h are monomeric helicases composed of three subdomains; subdomains 1 and 2 (red and orange in the inset of Fig 1) are RecA-like folds that are structurally conserved across all SF1 and SF2 helicases, whereas subdomain 3 (green) is unique to the NS3/NPH-II subfamily and contains some of the least conserved portions of the protein. In Fig 1, an adenosine triphosphate (ATP; purple) molecule is bound within the NTPase active site between subdomains 1 and 2. Also, an RNA substrate (blue) is bound within the RNA-binding cleft, separating subdomains 1 and 2 from subdomain 3. The 5′ terminus of the RNA is positioned at the top of the protein in Fig 1 and the ds/ss RNA junction is hypothesized to be just above this region of the protein. The NS3/NPH-II subfamily of SF2 helicases exhibit both RNA-stimulated NTPase activity and NTPase-dependent helicase activity [17–22]. These experimentally observed phenomena suggest that (1) the presence of RNA affects the NTPase active site, thereby activating the NTPase cycle and (2) this cycle is the source of free energy needed to perform work on the RNA (translocation and unwinding). In Fig 1, the enzymatic cycle for the NTPase function is depicted by four dynamic events: RNA is bound within the RNA-binding cleft and activates the NTPase cycle, NTP binds, NTP is hydrolyzed, and finally products (nucleotide diphosphate—NDP—and inorganic phosphate—H2PO4-, Pi) are released. To date, it is unclear which stage (s) of the cycle are responsible for the translocation and unwinding functions of NS3h. Furthermore, the biophysical couplings between NTPase and helicase active sites are still poorly understood [28]. One of the better studied Flaviviridae NS3h is that of the Hepatitis C virus (HCV; family: Flaviviridae hepacivirus) [29–40]. Utilizing both ensemble [29–35] and single molecule [36–38,41,42] techniques, studies have provided insights into the kinetic steps of the HCV NS3h translocation function. These studies, alongside crystallography studies of various Flaviviridae NS3h, suggest that the NS3 enzyme tracks along the phosphodiester backbone of the nucleic oligomer, unwinding one base-pair per hydrolysis event [35–37]. To explain these experimental results, various models describing the translocation mechanism have been reported, depicting NS3h as a Brownian [33–35] or backbone stepping motor [36,39–41] protein. These models envision the coupling between NTPase and helicase functions through different biophysical mechanisms, yet the models are not mutually exclusive and are limited in temporal and spatial resolution [43,44]. Luo et al. reported a set of crystal structures of the DENV NS3h in important protein-substrate complexes of the NTPase cycle (bolded text in Fig 1) [45]. From these structures, major allosteric influences of RNA-binding were seen in the NTPase active site. For example, Luo and coworkers noted that the presence of an RNA substrate shifts the carboxylate group of Glu285 (motif II) into a more catalytically relevant structure for the hydrolysis reaction. Mutation of the Glu285 residue abrogates NTPase and helicase activities [25]. These static structures have provided novel insights into RNA-induced protein structural changes yet provide limited insight into the NTPase cycle or translocation and unwinding functions of NS3h. Previous theoretical studies of helicases have focused on a broad range of enzymes such as PcrA (SF1) [46–49], transcription terminator Rho (SF5) [50], SV40 (SF3) [51], and various NS3h enzymes [52–56]. Of the theoretical studies on NS3h, Perez-Villa et al. reported microsecond-long molecular dynamics (MD) simulations of the HCV NS3h-ssRNA systems in the presence and absence of ATP and ADP. The reported simulations were used to interrogate the thermodynamics of these substrate states with various conformations of the NTPase active site [52]. While the reported results are of interest for NS3h, the authors provide limited insight into the molecular mechanisms at play during the NTPase cycle. Other theoretical studies of the NS3h enzyme are limited in timescales (tens to hundreds of ns of simulation), substrate states modeled, or spatial resolution (e. g. coarse grained elastic network model) [53–56]. Therefore, theoretical modeling of the NS3h enzyme has yet to elucidate further details about the structural and dynamic couplings within NS3h in light of the NTPase cycle. We report here a multiscale theoretical study of the DENV NS3h enzyme at each substrate state along the NTPase cycle. RNA-induced allostery on the NTPase active site is reported wherein the presence of an RNA substrate alters the positioning and dynamics of waters within the hydrolysis active site. Inspired by this observation, minimum energy electronic structure calculations are performed to investigate the energy landscape of the hydrolysis reaction. Additionally, investigations into NTPase substrate-induced allostery on the RNA-binding cleft suggest that NS3h interacts with RNA in a NTPase substrate-dependent manner. Umbrella sampling (US) simulations are performed to enhance the sampling of a proposed elementary step of the translocation mechanism observed during the unbiased simulations. Finally, analyses of the correlated motions between residues are used to identify allosteric pathways that connect the two active sites. It is through these pathways that we hypothesize that free energy released during the NTPase cycle is transduced to the RNA-binding cleft and utilized to perform work on the RNA. This study of the substrate states of DENV NS3h lays the foundation for further study of the NTPase cycle and marks the most complete picture of the molecular mechanism of the NS3 NTPase/helicase to date.
A subset of the crystal structures reported by Luo et al. [45] of the Dengue NS3h (serotype 4) are used as the initial structures for all-atom, explicit solvent MD simulations. Specifically, the binary complex of NS3h with a seven-residue ssRNA substrate (PDB ID: 2JLU) is used to model the ssRNA substrate state, while the ternary structures of ssRNA+ATP (2JLV), ssRNA+ADP+Pi (2JLY), and ssRNA+ADP (2JLZ) model the pre-hydrolysis, post-hydrolysis, and product release states of the NTPase cycle, respectively. The Apo (2JLQ) and ATP (2JLR) substrate states are also simulated and used as experimental controls for our investigation into allostery. The RNA-bound structures of DENV NS3h were crystalized as dimers of the protein [45]. For these systems, chain A of the structure is used as the starting conformation. Furthermore, the A conformers are chosen for residues with multiple side chain conformations. In all crystal structures with ATP substrates, the crystalized Mn2+ divalent cation is converted into a Mg2+. For the ATP crystal structure (2JLR), residues of the protease linker region were poorly resolved and so are transferred from the Apo (2JLQ) structure after aligning the neighboring amino acid backbones in both systems. All-atom, explicit solvent MD simulations are performed for the six substrate states of DENV NS3 and presented in Fig 1 (denoted Apo, ATP, ssRNA, ssRNA+ATP, ssRNA+ADP+Pi, and ssRNA+ADP). The simulations are performed using the GPU-enabled AMBER14 software [57], ff14SB [58] parameters for proteins, and ff99bsc0χ OL3 [59,60] parameters for RNA. Parameters for ATP [61], ADP [61], Pi (provided in Supplementary Information (SI); S2 File), and Mg2+ [62] are also used. For each system, the crystal structures are solvated in TIP3P water boxes with at least a 12 Å buffer between the protein and periodic images. Crystallographic waters are maintained. Sodium and chloride ions are added to neutralize charge and maintain a 0. 10 M ionic concentration. The Langevin dynamics thermostat and Monte Carlo barostat are used to maintain the systems at 300 K and 1 bar. Direct nonbonding interactions are calculated up to a 12 Å distance cutoff. The SHAKE algorithm is used to constrain covalent bonds that include hydrogen [63]. The particle-mesh Ewald method [64] is used to account for long-ranged electrostatic interactions. A 2 fs integration time step is used, with energies and positions written every 2 ps. The minimum amount of simulation performed for each system is one trajectory of 1. 5 μs, with the first 200 ns of simulation sacrificed to equilibration of the starting structures. Simulation of the ssRNA system is performed to 2 μs. For both the ATP and ssRNA+ATP systems, two 1. 5 μs simulations are performed. The total amount of unbiased simulation reported here on the described structures is 12. 5 μs. US simulations are performed to enhance sampling of a hypothesized elementary translocation event wherein the biased collective variable is the distance between the central carbon of the guanidinium group of Arg387 to the phosphorous atom of phosphate 4 in the RNA. These simulations are run for the ssRNA, ssRNA+ATP, ssRNA+ADP+Pi, and ssRNA+ADP systems, using the same protocol as the unbiased simulations with the addition of a bias. For each substrate state, a minimum of 22 sampling windows are simulated for 50 ns each with harmonic wells positioned every 0. 5 Å and ranging from 3. 50 to 14. 00 Å. Harmonic force constants are 20 kcal mol-1 Å-2. Further simulation and additional windows are run in regions of collective variable space with poor sampling. The weighted histogram analysis method (WHAM) [65] is used to analyze the results of these simulations, with bin sizes of 0. 1 Å. Bootstrapping is used to approximate error bars for the probability density and free energy plots shown. The total amount of biased simulations reported here is 5. 12 μs. Electronic structure calculations are performed at the ωB97X-D/6-31+G* level of theory [66] using the Guassian 09 version B. 01 program [67]. The ωB97X-D functional is chosen due to its broad applicability [68,69] and a recent study demonstrating its energetic accuracy for a variety of phosphate hydrolysis reactions [70]. The QM system is composed of a truncated ATP molecule (truncated to methyl triphosphate, MTP), functional groups of nine surrounding protein residues (Pro195, Gly196, Lys199, Glu285, Ala316, Gly414, Gln456, Arg460, and Arg463), a Mg2+ ion, and seven water molecules. The amino acids are truncated at various positions (more detail in S1 File) using hydrogen atoms. For each residue, the position of the terminal heavy atom is frozen to maintain the active site geometry. This yielded a total of 138 atoms in the QM calculations. These calculations are performed on active site conformations pulled from the unbiased MD simulations of the ssRNA+ATP and ATP substrate states, thereby investigating the influence of observed RNA structural allostery on the hydrolysis reaction mechanism and energy landscape. Frames used for the initial reactant state structures were selected by visualizing MD frames in which a lytic water is present. Through visual and RMSD analyses of such frames, a single frame was chosen to represent the population of catalytically relevant structures. The hydrolysis reaction is then monitored by optimizing the reactants (MTP+lytic water), products (MDP+HPO42−), and a single transition state (TS) in between. The initial TS and product state structures were created from the previous optimized structure. The minima are confirmed using a Hessian calculation. The TS is confirmed by examining the direction of the single imaginary frequency. Following geometry optimization, frequency calculations are performed to obtain gas-phase, zero-point energy corrected free energies for each active site conformation. Unless stated otherwise, analyses of MD trajectories are performed using Python 2. 7 and the MDAnalysis module (version 0. 15. 0) [71]. Matplotlib is used for plotting data [72]. VMD is used for visualization of trajectories and production of structural figures [73–75]. For each substrate state, a single frame from the trajectories is used when presenting structural details of the respective substrate state. Further information on choosing these “exemplar” structures is given in the S1 File. Additionally, details of all analyses performed can be found in the S1 File. All scripts for the analyses are available on Github (https: //github. com/mccullaghlab/DENV-NS3h).
To date, no biophysical explanation has been proposed for the 10 to 100-fold increase in NTPase turnover rate observed for DENV NS3h in the presence of RNA [22]. Crystallographic studies of the DENV NS3h structure have identified static structural allostery due to RNA binding [45], yet a dynamic picture and interpretation of these influences are still missing. In this section, comparisons of the simulations of the Apo, ATP, ssRNA, and ssRNA+ATP substrate states are used to depict structural rearrangements induced by RNA. These RNA-induced allosteries are observed to affect the positioning and dynamics of waters within the NTPase active site. These novel insights gained from the comparisons of the MD simulations inspire the reported electronic structure calculations of the reactant, transition, and product states of the hydrolysis reaction. In combination, these results demonstrate that the observed enhancement of NTPase activity originates from the RNA-induced destabilization of the lytic water. Experimental studies have shown that the NS3h helicase functions (translocation and unwinding) are NTPase dependent, yet it is unclear which equilibrium states and/or dynamic events of the NTPase cycle are the source of the necessary free energy for these functions [20,21]. All previously developed models describing these functions have deduced that the NTPase cycle drives conformational changes in the RNA-binding cleft, thereby cycling the protein-RNA interactions leading to unidirectional translocation and melting of the duplex/single stranded nucleic junction [33–36,39–41]. Yet, limited structural allostery attributed to the NTPase substrates (e. g. ATP, ADP, and Pi) is observed in the crystal structures of DENV NS3h [45]. Therefore, a subset of the MD simulations reported here (ssRNA, ssRNA+ATP, ssRNA+ADP+Pi, and ssRNA+ADP) is used to interrogate protein-RNA interactions as well as identify protein structural changes that have NTPase substrate-dependent behaviors. The current view of allosteric regulation focuses on signal transduction through complex, 3-dimensional networks, brought about by intrinsic structural and/or dynamic changes along pathways connecting two distal, non-overlapping active sites [96–98]. These allosteric pathways are described by coupled short-range, residue-residue interactions that lead to long-range correlations. In the previous two sections, RNA-induced and NTPase substrate-induced structural rearrangements have been presented. In this section, these allosteric structural changes are absorbed into a unified description of the allosteric pathways connecting the RNA-binding cleft with the NTPase active site. Dynamic network analyses, such as residue-residue correlations, have been used to identify allosteric pathways within proteins from simulation [96–100]. A growing body of literature has highlighted the functional importance of such pathways as well as the fundamental residue-residue interactions leading to their emergence [96–102]. We report here residue-residue distance correlation analyses that are used to identify the allosteric pathways present within the DENV NS3h protein. Focus is given to the motifs discussed in the previous sections (α2, motifs II and IVa) due to the observed structural rearrangements. Additionally, the correlation heat maps are used to identify segments of the protein that experience strong correlations with numerous other regions of the protein, such as motif V. While motif V does not experience substrate-induced structural rearrangements, the strong correlations between motif V and motifs in both the NTPase active site and RNA binding cleft are hypothesized to have functional importance in the signal transduction mechanism of allosteric regulation. Unlike the previous two sections, comparisons between substrate states (RNA-bound and NTPase substrate-bound) are not considered here. Instead, focus is given to the discussion of the residue-residue distance correlation analysis of the ssRNA+ATP substrate state. Through analyses of the reported simulations, molecular observables of RNA- and NTPase substrate-induced allostery were identified. Specifically, an RNA bound within the RNA-binding cleft affects the dynamics and positioning of water molecules within the NTPase active site. This allosteric influence is conferred from the RNA-binding cleft to the hydrolysis active site through structural rearrangements of Lβ3β4, α2, and motif II. These RNA-induced structural changes lead to an entropic destabilization of the NTPase active site as well as a direct destabilization of the lytic water. Inspired from these results, electronic structure calculations were used to investigate the energetics of NTP hydrolysis reaction. The energetic landscapes obtained from the DFT calculations demonstrate that RNA decreases the activation barrier as well as affects the mechanism of the hydrolysis reaction. Combining these results into a kinetic model allowed for the calculation of a theoretical RNA-stimulated NTPase activity enhancement factor of 150, which qualitatively matches the experimentally observed enhancement factor. Therefore, results from MD and DFT calculations provide novel, multiscale insight into the RNA-induced allosteric effects that stimulate the catalysis of the NTP hydrolysis reaction in DENV NS3h. Unlike RNA, the NTPase substrates are smaller perturbations to the NS3h structure and dynamics. Protein-RNA interaction energies were used to investigate the NTPase substrate-dependence of protein-RNA contacts in the unbiased MD simulations. From these analyses, the protein-RNA phosphodiester backbone interactions were observed to be NTPase substrate-dependent. The presence of the γ-phosphate (or Pi) of the NTPase substrate was observed to strengthen the protein-RNA contacts. Furthermore, the localized nonbonding interaction energies demonstrate a large shift in protein-RNA contacts, originating in part from the side chain conformational states of Arg387. Results from US simulations demonstrate that the Arg387 side chain conformational states exemplify NTPase substrate-dependent protein-RNA interactions. With the purview of the NTPase cycle, transitions between conformational states leads to 3′ to 5′ translocation. Therefore, we hypothesize that the transition between Arg387 side chain conformations is an elementary step in the unidirectional translocation mechanism of NS3h along the phosphodiester backbone of RNA. Finally, consideration of these allosteric effects independent of one another provides an incomplete picture of the biophysics of the NS3h protein. Residue-residue correlation analyses were used to identify structural regions of the protein that experienced correlated motions with other regions. These analyses were used to describe the allosteric pathways that connect α2 with motifs II and IVa. The short-range, residue-residue interactions were presented that connect the RNA-binding cleft to the NTPase active site. Furthermore, the correlation heat maps allow for identification of regions of the protein that experience strong correlated motions with numerous other regions. Motif V is one such example, where the segment of 13 residues has strong coupled motions with seven other motifs in subdomains 1 and 2. This highly correlated nature suggests that motif V functions as a centralized communication hub that connects distal portions of the protein structure. Complete modeling of a revolution of the NTPase cycle in the DENV NS3h presents a significant challenge for current computational methodologies. Rather, we have divided the cycle into equilibrium substrate states and dynamic events where the protein transitions from one substrate state to another. The simulations reported have modeled the important NTPase cycle substrate states, leading to novel insights into the function and underlying biophysics of the DENV NS3h enzyme with focus given to the allosteric connections between the RNA-binding cleft and NTPase active site. We hypothesize that the observed allosteric effects and pathways have important roles in the transduction of energy from one active site to the other during the dynamic events of the NTPase cycle. Therefore, the results reported have laid an initial foundation for theoretical investigations into the dynamic events of the NTPase cycle. Beyond further theoretical modeling of NS3h, transverse relaxation-optimized spectroscopy (TROSY) NMR, mutational biochemical studies, and targeted small molecule binding experiments can be envisioned to test the hypothesis and results presented. Nuclear magnetic resonance has been previously used to study dynamics within isolated HCV NS3 helicase subdomains [103–105], but the size of the full dengue NS3h domain is too large for traditional NMR approaches due to line width increasing with increased molecular mass. However, TROSY NMR has been developed that may allow for experimental monitoring of fast NS3h dynamics [106]. We anticipate that perturbation of the wild-type structure or dynamics of the allosteric pathways in NS3h will lead to abrogation of NTPase and/or helicase functions, and are currently developing assays to test this hypothesis with dengue NS3h. Residues active in the allosteric pathways are viable targets for mutational studies where varying a specific amino acid residue is expected to alter the short-range, residue-residue interactions and lead to a destabilization of the pathway connecting the two active sites. This is hypothesized to result in reductions in enzymatic activity and would be observable in our biochemical assays. Additionally, these pathways are viable targets for theoretical and experimental small molecule drug docking experiments with focus given to molecules that disrupt the residue-residue interactions along the pathway. Co-crystallization of specific conformation-binding molecules may lock NS3h into transition-state conformations that can help verify our computational studies. Molecular candidates also have the potential for inhibiting NS3h function during replication and being specific to the NS3/NPH-II subfamily of SF2 helicases.
Non-structural protein 3 (NS3) is a Flaviviridae (e. g. Hepatitis C, dengue, and Zika viruses) helicase that unwinds double stranded RNA while translocating along the nucleic polymer during viral genome replication. As a member of superfamily 2 (SF2) helicases, NS3 utilizes the free energy of nucleotide triphosphate (NTP) binding, hydrolysis, and product unbinding to perform its functions. While much is known about SF2 helicases, the pathways and mechanisms through which free energy is transduced between the NTP hydrolysis active site and RNA binding cleft remains elusive. Here we present a multiscale computational study to characterize the allosteric effects induced by the RNA and NTPase substrates (ATP, ADP, and Pi) as well as the pathways of short-range, residue-residue interactions that connect the two active sites. Results from this body of molecular dynamics simulations and electronic structure calculations are highlighted in context to the NTPase enzymatic cycle, allowing for development of testable hypotheses for validation of these simulations. Our insights, therefore, provide novel details about the biophysics of NS3 and guide the next generation of experimental studies.
allosteric regulation
crystal structure
protein interactions
enzymes
condensed matter physics
enzymology
crystallography
thermodynamics
enzyme chemistry
rna structure
solid state physics
proteins
enzyme regulation
chemistry
molecular biology
free energy
physics
biochemistry
rna
biochemical simulations
hydrolysis
helicases
nucleic acids
biology and life sciences
chemical reactions
physical sciences
computational biology
macromolecular structure analysis
Allostery in the dengue virus NS3 helicase: Insights into the NTPase cycle from molecular simulations