Motif-VI loop acts as a nucleotide valve in the West Nile Virus NS3 Helicase.

The Orthoflavivirus NS3 helicase (NS3h) is crucial in virus replication, representing a potential drug target for pathogenesis. NS3h utilizes nucleotide triphosphate (ATP) for hydrolysis energy to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. Intermediate states along the ATP hydrolysis cycle and conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. Extensive molecular dynamics simulations of West Nile virus NS3h+ssRNA in the apo, ATP, ADP+Pi and ADP bound states were used to model the conformational ensembles along this cycle. Energetic and structural clustering analyses depict a clear trend of differential enthalpic affinity of NS3h with ADP, demonstrating a probable mechanism of hydrolysis turnover regulated by the motif-VI loop (MVIL). Based on these results, MVIL mutants (D471L, D471N and D471E) were found to have a substantial reduction in ATPase activity and RNA replication compared to the wild-type. Simulations of the mutants in the apo state indicate a shift in MVIL populations favoring either a closed or open 'valve' conformation, affecting ATP entry or stabilization, respectively. Combining our molecular modeling with experimental evidence highlights a conformation-dependent role for MVIL as a 'valve' for the ATP-pocket, presenting a promising target for antiviral development.

Role of ATP Hydrolysis and Product Release in the Translocation Mechanism of SARS-CoV-2 NSP13.

In response to the emergence of COVID-19, caused by SARS-CoV-2, there has been a growing interest in understanding the functional mechanisms of the viral proteins to aid in the development of new therapeutics. Nonstructural protein 13 (nsp13) helicase is an attractive target for antivirals because it is essential for viral replication and has a low mutation rate, yet the structural mechanisms by which this enzyme binds and hydrolyzes ATP to cause unidirectional RNA translocation remain elusive. Using Gaussian accelerated molecular dynamics (GaMD), we generated comprehensive conformational ensembles of all substrate states along the ATP-dependent cycle. Shape-GMM clustering of the protein yields four protein conformations that describe an opening and closing of both the ATP pocket and the RNA cleft that is achieved through a combination of conformational selection and induction along the ATP hydrolysis cycle. Furthermore, three protein-RNA conformations are observed that implicate motifs Ia, IV, and V as playing a pivotal role in an ATP-dependent inchworm translocation mechanism. Finally, based on a linear discriminant analysis of protein conformations, we identify L405 as a pivotal residue for the opening and closing mechanism and propose a L405D mutation as a way to disrupt translocation. This research enhances our understanding of nsp13's role in viral replication and could contribute to the development of antiviral strategies.

Conserved motifs in the flavivirus NS3 RNA helicase enzyme.

Flaviviruses are a major health concern because over half of the world population is at risk of infection and there are very few antiviral therapeutics to treat diseases resulting from infection. Replication is an essential part of the flavivirus survival. One of the viral proteins, NS3 helicase, is critical for unwinding the double stranded RNA intermediate during flaviviral replication. The helicase performs the unwinding of the viral RNA intermediate structure in an ATP-dependent manner. NS3 helicase is a member of the Viral/DEAH-like subfamily of the superfamily 2 helicase containing eight highly conserved structural motifs (I, Ia, II, III, IV, IVa, V, and VI) localized between the ATP-binding and RNA-binding pockets. Of these structural motifs only three are well characterized for function in flaviviruses (I, II, and VI). The roles of the other structural motifs are not well understood for NS3 helicase function, but comparison of NS3 with other superfamily 2 helicases within the viral/DEAH-like, DEAH/RHA, and DEAD-box subfamilies can be used to elucidate the roles of these structural motifs in the flavivirus NS3 helicase. This review aims to summarize the role of each conserved structural motif within flavivirus NS3 in RNA helicase function. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.

Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase.

The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but nonstructural protein 13 (nsp13) stands out as a highly conserved and yet understudied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP-dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound, which presents a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 µs of a Gaussian-accelerated molecular dynamics simulation (at least 6 µs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A Gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key structural states of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inchworm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV, and V and suggest a power stroke-like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.

Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft.

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

Molecular Dynamics Simulations of 2-Aminopurine-Labeled Dinucleoside Monophosphates Reveal Multiscale Stacking Kinetics.

Molecular dynamics (MD) simulations of 2-aminopurine (2Ap)-labeled DNA dinucleoside monophosphates (DNMPs) were performed to investigate the hypothesis that base stacking dynamics occur on timescales sufficiently rapid to influence the emission signals measured in time-resolved fluorescence experiments. Analysis of multiple microsecond-length trajectories shows that the DNMPs sample all four coplanar stacking motifs. In addition, three metastable unstacked conformations are detected. A hidden Markov-state model (HMSM) was applied to the simulations to estimate transition rates between the stacked and unstacked states. Transitions between different stacked states generally occur at higher rates when the number of nucleobase faces requiring desolvation is minimized. Time constants for structural relaxation range between 1.6 and 25 ns, suggesting that emission from photoexcited 2Ap, which has an excited-state lifetime of 10 ns, is sensitive to base stacking kinetics. A master equation model for the excited-state population of 2Ap predicts multiexponential emission decays that reproduce the sub-10 ns emission decay lifetimes and amplitudes seen in experiments. Combining MD simulations with HMSM analysis is a powerful way to understand the dynamics that influence 2Ap excited-state relaxation and represents an important step toward using observed emission signals to validate MD simulations.

Initial Aggregation and Ordering Mechanism of Diphenylalanine from Microsecond All-Atom Molecular Dynamics Simulations.

Self-assembled diphenylalanine (FF) nanostructures have recently been demonstrated to be interesting materials for antibacterial and anticancer applications. These applications, among others, seek to take advantage of the high-order and resulting appealing physical properties of FF nanostructures by modifying the peptide in some way to achieve specific functionality. To rationally design modifications to the dipeptide that allow for this behavior, the driving forces of FF self-assembly must be understood. Molecular simulations have been utilized to assess these properties but have yielded conflicting conclusions due to inconsistencies in models chosen as well as the lack of quantitative analyses on the specific driving forces. Here, we present an all-atom explicit solvent molecular dynamics-based study on different length scales of FF aggregation. We utilize a free energy decomposition analysis as well as a dimer cluster analysis to identify the initial aggregation driving force to be FF intermolecular electrostatics, whereas solvent-mediated forces drive crystal growth. These data are consistent with the hypothesis that all hydrophobic dipeptides will have a similar initial aggregation mechanism until a critical aggregate size is reached, at which point crystallization occurs and subsequent crystal growth is dominated by solvent-mediated forces. We demonstrate that this proposed mechanism is testable by infrared spectroscopy focusing on the blueshift of the amide I peak as well as the ordering of the carboxylate peak.

Allostery in the dengue virus NS3 helicase: Insights into the NTPase cycle from molecular simulations.

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.

Unraveling the mystery of ATP hydrolysis in actin filaments.

Actin performs its myriad cellular functions by the growth and disassembly of its filamentous form. The hydrolysis of ATP in the actin filament has been shown to modulate properties of the filament, thus making it a pivotal regulator of the actin life cycle. Actin has evolved to selectively hydrolyze ATP in the filamentous form, F-actin, with an experimentally observed rate increase over the monomeric form, G-actin, of 4.3 × 10(4). The cause of this dramatic increase in rate is investigated in this paper using extensive QM/MM simulations of both G- and F-actin. To compute the free energy of hydrolysis in both systems, metadynamics is employed along two collective variables chosen to describe the reaction coordinates of hydrolysis. F-actin is modeled as a monomer with restraints applied to coarse-grained variables enforced to keep it in a filament-like conformation. The simulations reveal a barrier height reduction for ATP hydrolysis in F-actin as compared to G-actin of 8 ± 1 kcal/mol, in good agreement with the experimentally measured barrier height reduction of 7 ± 1 kcal/mol. The barrier height reduction is influenced by an enhanced rotational diffusion of water in F-actin as compared to G-actin and shorter water wires between Asp154 and the nucleophilic water in F-actin, leading to more rapid proton transport.

Modeling self-assembly processes driven by nonbonded interactions in soft materials.

This Centennial Feature Article provides an overview of research in the general area of self-assembly modeling, with particular emphasis on the self-assembly of molecules into soft nanoscale structures where the driving force for assembly is provided by nonbonded interactions (hydrogen bonds and electrostatics). The models have been developed at many different levels of theory, going all the way from simple analytical models of packing effects to atomistic descriptions using molecular dynamics methods. In between these limits are mean-field and coarse-grained models, including models for DNA, peptides, and lipids that can be used to describe the assembly of hybrid (amphiphilic) materials. Several recent applications to specific systems are discussed, including the description of peptide amphiphile assembly to make cylindrical micelles, the assembly and melting of DNA hairpins, the use of DNA tethers to assemble nanoparticles into aggregates and crystalline structures, and the use of coarse-grained lipid models to make lamellar and high-curvature phases. These examples demonstrate the difficulties associated with brute force atomistic methods, and they also show the opportunities (but uncertainties and ambiguities) associated with simpler models such as coarse-grained models. The examples also demonstrate the usefulness of successful modeling methods in the design of new materials, including an understanding of the relationship between structure and function.