Biology subjects

McCullagh, M.

Publications and source records attributed to McCullagh, M..

13 recordsLinked to original sources

bioRxiv2026

Mechanochemical Decoupling of ATP Hydrolysis and RNA Translocation in SARS-CoV-2 nsp13 by the L405D Mutation

SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved helicase that couples ATP hydrolysis to RNA translocation through long-range allosteric communication between its ATPase and RNA-binding domains. In prior work, we identified L405 as a key regulator of interdomain motions and proposed that the L405D mutation would disrupt this coupling by perturbing conformational translocations required for translocation [J. Phys. Chem. B 2024 v128 492-503]. Subsequent experiments confirmed that L405D attenuates helicase activity while largely preserving ATPase activity, implicating a breakdown in ATP-to-RNA coupling [J. Biol. Chem. 2026 v302 111198]. Here, we provide a data-driven explanation for this decoupling by combining Gaussian accelerated molecular dynamics (GaMD) simulations with Shape-GMM clustering and linear discriminant analysis. Whereas wild-type nsp13 exhibits both conformational selection and induction, L405D collapses the conformational landscape to operate predominantly through selection, eliminating ATP-induced structural transitions required for efficient catalytic cycling. This loss of induction traps the ATP-binding pocket in a mid-open conformation, impairing product release and reducing ATP turnover, while simultaneously disrupting coordinated motif-RNA interactions required for inchworm translocation. These findings establish that mutation-induced reshaping of conformational ensembles can modulate access to reaction-competent states, providing a general framework for understanding how targeted mutations disrupt catalytic function through allosteric ensemble remodeling in motor proteins.

biophysics↗

bioRxiv2026

The Impact of Glycosylation on the Conformational Ensembles of β-, δ-, and γ-Sarcoglycans

Glycosylation is a pivotal post-translational modification that influences protein folding, stability, and interactions, with direct implications for muscular dystrophy pathogenesis and emerging gene therapies. Sarcoglycans (SGs), {beta}-,{delta} -,{gamma} -, and - subunits of the dystrophin-glycoprotein complex (DGC), contain essential N-linked glycosylation sites, and mutations that disrupt glycan attachment, destabilize the complex and cause limb-girdle muscular dystrophy. Yet, the structural consequences of SG glycosylation remain poorly defined due to the absence of experimental sarcoglycan complex structures. Here, we use homology modeling, AlphaFold predictions, and allatom molecular dynamics simulations to probe how N-linked glycans reshape the conformational ensembles of {beta}-,{delta} -, and{gamma} -SG monomers and the {beta}-{delta}-{gamma} heterotrimer core. We find that glycosylation increases flexibility and conformational heterogeneity in isolated monomers but reinforces a compact, stabilized architecture in the heterotrimer. Contact map and clustering analyses show that glycans redistribute local residue interactions while preserving global trimer organization, suggesting a context-dependent role in destabilizing monomers yet reinforcing complex stability. These findings provide the first atomistic insight into how glycosylation primes sarcoglycans for assembly and may explain why mutations at glycosylation sites disrupt complex integrity and drive muscular dystrophy phenotypes.

biophysics↗

bioRxiv2025

Tutorial on quantifying and sampling biomolecular ensembles withShapeGMM

Here we present a detailed workflow for clustering and enhanced sampling of biomolecular conformations using the ShapeGMM methodology. This approach fits a probabilistic model of biomolecular conformations rooted in the idea that the free energy can expressed in terms of local fluctuations in atomic positions around metastable states. We demonstrate using a single model system how to generate and fit equilibrium molecular dynamics simulation data. We then show how to use the resulting model to generate a reaction coordinate between two states, how to sample along that coordinate using Metadynamics using our size-and-shape PLUMED module, and how to cluster those biased conformations to give a refined equilibrium ShapeGMM model.

biophysics↗

bioRxiv2025

Allosteric Regulation of RNA Affinity by Motif V-VI Coupling in West Nile Virus NS3 Helicase

The rise of flaviviral diseases, including West Nile virus (WNV), presents a growing threat to global public health and underscores the urgent need for new therapeutic strategies. The non-structural protein 3 helicase (NS3h) of the Orthoflavivirus genus, including WNV, is essential for viral replication and a promising antiviral target. Previously [Roy et al., Nucleic Acids Research, 52, 13, 2024, 7447-7464], we showed that the motif VI loop (VIL) in WNV NS3h functions as a nucleotide valve, regulating ADP affinity during hydrolysis. In this study, we uncover an ATP-dependent coupling between nucleotide affinity at motif VIL and RNA affinity at motifs IVa and V, suggesting a coordinated mechanism of ssRNA translocation. Using microsecond-scale all-atom molecular dynamics simulations of hydrolysis-cycle intermediates, we find that key VIL residues (R461, R464) correlate strongly with RNA phosphate affinity of motif V. Structural analyses reveal an ATP-sensitive interaction between E413 (motif V) and R461 (motif VIL) that modulates the conformation of the motif V 310-helix, thereby influencing RNA binding. This dynamic interaction is lost in catalytically deficient VIL mutants, which have been experimentally shown to impair hydrolysis and attenuate viral replication. These findings provide mechanistic insights into NS3h function and identify new opportunities for structure-based antiviral design.

biophysics↗

bioRxiv2025

Improved data-driven collective variables for biased sampling through iteration on biased data

Our ability to efficiently sample conformational transitions between two known states of a biomolecule using collective variable (CV) based sampling depends strongly on the choice of the CV. We previously reported a data-driven approach to clustering biomolecular configurations with a probabilistic clustering model termed ShapeGMM. ShapeGMM is a Gaussian Mixture Model in cartesian coordinates, with means and covariances in each cluster representing the harmonic approximation to the conformational ensemble around a metastable state. We subsequently showed that Linear Discriminant Analysis on positions (posLDA) is a good reaction coordinate to characterize the transition between two of these states, and moreover can be biased to produce transitions between the states using Metadynamics-like approaches. However, the quality of these LDA coordinates depends on the amount of data used to characterize the states, and here we demonstrate the ability to systematically improve them using enhanced sampling data. Specifically, we demonstrate that improved CVs for sampling can be generated by iteratively performing biased sampling along a posLDA coordinate and then generating a new shapeGMM model from biased data in the previous iteration. The new coordinates derived from our iterative approach show a substantial improvement in being able to induce transitions between metastable states, and to converge a free energy surface.

biophysics↗

bioRxiv2024

Quantifying Unbiased Conformational Ensembles from Biased Simulations Using ShapeGMM

Quantifying the conformational ensembles of biomolecules is fundamental to describing mechanisms of processes such as ligand binding and allosteric regulation. Accurate quantification of these ensembles remains a challenge for all but the simplest molecules. One such challenge is insufficient sampling which enhanced sampling approaches, such as metadynamics, were designed to overcome; however, the non-uniform frame weights that result from many of these approaches present an additional challenge to ensemble quantification techniques such as Markov State Modeling or structural clustering. Here, we present rigorous inclusion of non-uniform frame weights into a structural clustering method entitled shapeGMM. The shapeGMM method fits a Gaussian mixture model to particle positions, and here we advance that approach by incorporating nonuniform frame weights in the estimates of all parameters of the model. The resulting models are high dimensional probability densities for the unbiased systems from which we can compute important thermodynamic properties such as relative free energies and configurational entropy. The accuracy of this approach is demonstrated by the quantitative agreement between GMMs computed by Hamiltonian reweighting and direct simulation of a coarse-grained helix model system. Furthermore, the relative free energy computed from a high dimensional probability density of alanine dipeptide reweighted from a metadynamics simulation quantitatively reproduces the metadynamics free energy in the basins. Finally, the method identifies hidden structures along the actin globular to filamentous-like structural transition from a metadynamics simulation on a linear discriminant analysis coordinate trained on GMM states, demonstrating the broad applicability of combining our prior and new methods, and illustrating how structural clustering of biased data can lead to biophysical insight. Combined, these results demonstrate that frame-weighted shapeGMM is a powerful approach to quantify biomolecular ensembles from biased simulations.

biophysics↗

bioRxiv2023

Motif-VI Loop Acts as a Nucleotide Valve in the West Nile Virus NS3 Helicase

The flavivirus NS3 helicase (NS3h), a highly conserved protein, plays a pivotal role in virus replication and thus represents a potential drug target for flavivirus pathogenesis. NS3h utilizes nucleotide triphosphate, such as ATP, for hydrolysis energy (ATPase) to translocate on single-stranded nucleic acids, which is an important step in the unwinding of double-stranded nucleic acids. The intermediate states along the ATP binding and hydrolysis cycle, as well as the conformational changes between these states, represent important yet difficult-to-identify targets for potential inhibitors. We use extensive molecular dynamics simulations of apo, ATP, ADP+Pi, and ADP bound to WNV NS3h+ssRNA to model the conformational ensembles along this cycle. Energetic and structural clustering analyses on these trajectories 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). These findings were experimentally corroborated using viral replicons encoding three mutations at the D471 position. Replication assays using these mutants demonstrated a substantial reduction in viral replication compared to the wild-type. Molecular simulations of the D471 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.

biochemistry↗

bioRxiv2023

The 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. Non-structural 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 a comprehensive conformational ensemble of all substrate states along the ATP-dependent cycle. ShapeGMM clustering of the protein yields four protein conformations that describe an opening and closing of both the ATP pocket and RNA cleft. This opening and closing is achieved through a combination of conformational selection and induction along the ATP 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 of testing our proposed mechanism. This research enhances our understanding of nsp13s role in viral replication and could contribute to the development of antiviral strategies. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/560057v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@17afdfeorg.highwire.dtl.DTLVardef@1b95faborg.highwire.dtl.DTLVardef@137d575org.highwire.dtl.DTLVardef@9f3593_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

The 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 non-structural protein 13 (nsp13) stands out as a highly conserved and yet under studied 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 presenting 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 s of Gaussian accelerated molecular dynamics simulation (at least 6 s 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 aspects of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inch-worm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV and V and suggest a powerstroke-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.

biophysics↗

bioRxiv2020

A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes

The unwinding of double-stranded RNA intermediates is critical for replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that Motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in Motif V exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of Motif V in viral replication using T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo. We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection, and increased cytopathogenicity in cell culture as compared to WT Kunjin. Similarly, decreased infection rates in surviving S411A-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these data indicate that NS3 Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus. IMPORTANCEKunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years demonstrating they remain a global threat. Currently, there are no approved anti-viral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigate the role of the Kunjin virus helicase on infection in cell culture and in vivo. This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

microbiology↗

bioRxiv2020

RNA-dependent structures of the RNA-binding loop in the flavivirus NS3 helicase

The flavivirus NS3 protein is a helicase that has pivotal functions during the viral genome replication process, where it unwinds double-stranded RNA and translocates along the nucleic acid polymer in a nucleoside triphosphate hydrolysis-dependent mechanism. An increased interest in this enzyme as a potential target for development of antiviral therapeutics was sparked by the 2015 Zika virus epidemic in the Americas. Crystallographic and computational studies of the flavivirus NS3 helicase have identified the RNA-binding loop as an interesting structural element, which may function as an origin for the RNA-enhanced NTPase activity observed for this family of helicases. Microsecond-long unbiased molecular dynamics as well as extensive replica exchange umbrella sampling simulations of the Zika NS3 helicase have been performed to investigate the RNA-dependence of this loops structural conformations. Specifically, the effect of the bound single-stranded RNA (ssRNA) oligomer on the putative "open" and "closed" conformations of this loop are studied. In the Apo substrate state, the two structures are nearly isoergonic ({Delta}GO[->]C = -0.22 kcal mol-1), explaining the structural ambiguity observed in Apo NS3h crystal structures. The bound ssRNA is seen to stabilize the "open" conformation ({Delta}GO[->]C = 1.97 kcal mol-1) through direct protein-RNA interactions at the top of the loop. Interestingly, a small ssRNA oligomer bound over 13 [A] away from the loop is seen to affect the free energy surface to favor the "open" structure while minimizing barriers between the two states. The mechanism of the transition between "open" and "closed" states is characterized as are residues of importance for the RNA-binding loop structures. From these results, the loop is hypothesized to be a viable region in the protein for targeted small-molecule inhibition and mutagenesis studies, where stabilization of the "closed" RNA-binding loop will negatively impact RNA-binding and the RNA-enhanced NTPase activity.

biophysics↗

bioRxiv2019

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

The unwinding of double-stranded RNA intermediates is a critical component for the replication of flavivirus RNA genomes. This function is achieved by the C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 is known to require 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 published by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. In order to investigate the role of Motif V in this process, a study combining molecular dynamics, biochemistry and virology has been employed. Mutations of Motif V were tested in both replicon and recombinant protein systems to investigate viral genome replication, RNA binding affinity, ATP hydrolysis activity and helicase unwinding activity. Using these analyses, we found that T407A and S411A in Motif V demonstrated increased turnover rates, suggesting that the mutations causes the helicase to unwind dsRNA more quickly than WT. Additionally, simulations of each mutant were used 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 data help define the linkage between ATP hydrolysis and helicase activity within NS3 and provide insight into the biophysical mechanisms for ATPase driven NS3 helicase function.

biochemistry↗

bioRxiv2019

The Effective Coarse-grained Hessian Dictates Allostery in IGPS

The long-ranged coupling between residues that gives rise to allostery in a protein is built up from short-ranged physical interactions. Computational tools used to predict this coupling and its functional relevance have relied on the application of graph theoretical metrics to residue-level correlations measured from all-atom molecular dynamics (aaMD) simulations. The short-ranged interactions that yield these long-ranged residue-level correlations are quantified by the effective coarse-grained Hessian. Here we compute an effective harmonic coarse-grained Hessian from aaMD simulations of a benchmark allosteric protein, IGPS, and demonstrate the improved locality of this graph Laplacian over two other connectivity matrices. Additionally, two centrality metrics are developed that indicate the direct and indirect importance of each residue at producing the covariance between the effector binding pocket and the active site. The residue importance indicated by these two metrics is corroborated by previous mutagenesis experiments and leads to unique functional insights; in contrast to previous computational analyses, our results suggest that fP76-hK181 is the most important contact for conveying direct allosteric paths across the HisF-HisH interface. The connectivity around fD98 is found to be important at affecting allostery through indirect means.

biophysics↗