Quantifying Unbiased Conformational Ensembles from Biased Simulations Using ShapeGMM.

Quantifying the conformational ensembles of biomolecules is fundamental to describing mechanisms of processes such as protein folding, interconversion between folded states, ligand binding, and allosteric regulation. Accurate quantification of these ensembles remains a challenge for conventional molecular simulations of all but the simplest molecules due to insufficient sampling. Enhanced sampling approaches, such as metadynamics, were designed to overcome this challenge; however, the nonuniform 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 nonuniform frame weights into a structural clustering method entitled shapeGMM. The result of frame-weighted shapeGMM is a high dimensional probability density and generative model for the unbiased system 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 shapeGMM probability density of alanine dipeptide reweighted from a metadynamics simulation quantitatively reproduces the underlying 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, 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 quantifying biomolecular ensembles from biased simulations.

What is allosteric regulation? Exploring the exceptions that prove the rule!

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.

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.

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.

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 nsp13's role in viral replication and could contribute to the development of antiviral strategies.

Reaction Coordinates for Conformational Transitions Using Linear Discriminant Analysis on Positions.

In this work, we demonstrate that Linear Discriminant Analysis (LDA) applied to atomic positions in two different states of a biomolecule produces a good reaction coordinate between those two states. Atomic coordinates of a macromolecule are a direct representation of a macromolecular configuration, and yet, they are not used in enhanced sampling studies due to a lack of rotational and translational invariance. We resolve this issue using the technique of our prior work, whereby a molecular configuration is considered a member of an equivalence class in size-and-shape space, which is the set of all configurations that can be translated and rotated to a single point within a reference multivariate Gaussian distribution characterizing a single molecular state. The reaction coordinates produced by LDA applied to positions are shown to be good reaction coordinates both in terms of characterizing the transition between two states of a system within a long molecular dynamics (MD) simulation and also ones that allow us to readily produce free energy estimates along that reaction coordinate using enhanced sampling MD techniques.

Modeling Catalysis in Allosteric Enzymes: Capturing Conformational Consequences.

Greater understanding of enzymatic mechanisms aids the discovery of new targets for biologics, the development of biocatalytic transformations, and de novo enzyme design. Methods using quantum mechanical (QM) potentials, such as Density Functional Theory (DFT), have enabled complex multistep enzymatic mechanisms to be studied, often in quantitative detail. Nevertheless, the dynamic interconversion of enzyme conformations between active and inactive catalytic forms, involving length- and timescales inaccessible to QM treatments, presents a formidable challenge for the development of computational models for allosterically modulated enzymes. We present an overview of the key concepts underlying multistate models of enzyme catalysis, enzyme allostery, and the challenge that large-scale conformational changes pose for methods using QM, QM/MM, and MM potentials. Structural clustering is highlighted as a valuable approach to bridge molecular dynamics conformational sampling of MM potentials and quantum chemical cluster models of catalysis. Particularly relevant to this discussion is structural allostery, which serves as the exemplar of conformational consequences. Here, a well-characterized allosteric enzyme, Imidazole Glycerol Phosphate Synthase (IGPS), is used to showcase the importance of multiple conformations and guide a new direction for qualitative understanding and quantitative modeling in enzyme catalysis.

Size-and-Shape Space Gaussian Mixture Models for Structural Clustering of Molecular Dynamics Trajectories.

Determining the optimal number and identity of structural clusters from an ensemble of molecular configurations continues to be a challenge. Recent structural clustering methods have focused on the use of internal coordinates due to the innate rotational and translational invariance of these features. The vast number of possible internal coordinates necessitates a feature space supervision step to make clustering tractable but yields a protocol that can be system type-specific. Particle positions offer an appealing alternative to internal coordinates but suffer from a lack of rotational and translational invariance, as well as a perceived insensitivity to regions of structural dissimilarity. Here, we present a method, denoted shape-GMM, that overcomes the shortcomings of particle positions using a weighted maximum likelihood alignment procedure. This alignment strategy is then built into an expectation maximization Gaussian mixture model (GMM) procedure to capture metastable states in the free-energy landscape. The resulting algorithm distinguishes between a variety of different structures, including those indistinguishable by root-mean-square displacement and pairwise distances, as demonstrated on several model systems. Shape-GMM results on an extensive simulation of the fast-folding HP35 Nle/Nle mutant protein support a four-state folding/unfolding mechanism, which is consistent with previous experimental results and provides kinetic details comparable to previous state-of-the art clustering approaches, as measured by the VAMP-2 score. Currently, training of shape-GMMs is recommended for systems (or subsystems) that can be represented by ≲200 particles and ≲100k configurations to estimate high-dimensional covariance matrices and balance computational expense. Once a shape-GMM is trained, it can be used to predict the cluster identities of millions of configurations.

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.

Implicit Solvation Using the Superposition Approximation (IS-SPA): Extension to Peptides in a Polar Solvent.

Efficient, accurate, and adaptable implicit solvent models remain a significant challenge in the field of molecular simulation. A recent implicit solvent model, IS-SPA, based on approximating the mean solvent force using the superposition approximation, provides a platform to achieve these goals. IS-SPA was originally developed to handle nonpolar solutes in a polar solvent and did not accurately capture polar solvation. Here, we demonstrate that IS-SPA can accurately capture polar solvation by incorporating solvent orientation and accounting for the contributions from long ranged electrostatics. Solvent orientation is approximated as that of an ideal dipole aligned in a mean electrostatic field and an analytic form of the long ranged electrostatics is derived. Parameters for the model are calculated from explicit solvent simulations of an isolated atom or molecule and include atom-based solvent densities, mean electric field functions, radially symmetric averaged Lennard-Jones forces, and multipoles of the explicit solvent model. Using these parameters, IS-SPA accounts for asymmetry of charge solvation and reproduces the explicit solvent potential of mean force of dimerization of two oppositely charged Lennard-Jones spheres in chloroform with high fidelity. Additionally, the model more accurately captures the effect of explicit solvent on the monomer and dimer configurations of alanine dipeptide in chloroform than a generalized Born or constant density dielectric model. The current version of the algorithm is expected to outperform explicit solvent simulations for aggregation of small peptides at concentrations below 150 mM, well above the typical experimental concentrations for these materials.

NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4.

Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix-helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity.

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

The unwinding of double-stranded RNA intermediates is critical for the 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 West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) 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 compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral 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.IMPORTANCE Kunjin 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 that they remain a global threat. Currently, there are no approved antiviral 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 investigated 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.

Residue-Level Allostery Propagates through the Effective Coarse-Grained Hessian.

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 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 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.

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. Crystallographic and computational studies of the flavivirus NS3 helicase have identified the RNA-binding loop as an interesting structural element that may function as a component of the RNA-enhanced NTPase activity observed for this family of helicases. Microsecond-long unbiased molecular dynamics and extensive replica exchange umbrella sampling simulations of the Zika NS3 helicase have been performed to investigate the RNA dependence of this loop's structural conformations. Specifically, the effect of the bound single-stranded RNA (ssRNA) oligomer on the putative "open" and "closed" conformations of this loop is studied. In the Apo substrate state, the two loop conformations are nearly isoergonic (ΔAO→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 (ΔAO→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 Å away from the loop is seen to affect the free energy surface to favor the "open" structure, while minimizing barriers between the two states. Both the mechanism of the "open" to "closed" transition and important residues of the RNA-binding loop structures are characterized. From these results, point mutations that are hypothesized to stabilize the "closed" RNA-binding loop and negatively impact RNA-binding and the RNA-enhanced NTPase activity are posited.

The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX)4 and Coumarin-(RXDX)4 Conjugate ß-Sheets.

pH-Switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X = Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX)4 systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X = Ala are found to be unstable at both pHs, while conjugates with X = Val, Leu, Ile, and Phe are found to form stable ß-sheets at least at neutral pH. The coumarin-(RFDF)4 conjugate is found to have the largest relative entropy value of 0.884 ± 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF)4 containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.

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.

Characterization of the Search Complex and Recognition Mechanism of the AlkD-DNA Glycosylase.

DNA damage is a routine problem for cells, and pathways such as base excision repair have evolved to protect the genome by using DNA glycosylases to first recognize and excise lesions. The search mechanism of these enzymes is of particular interest due to the seemingly intractable problem of probing the billions of base pairs in the genome for potential damage. It has been hypothesized that glycosylases form multiple protein-DNA conformational states to efficiently search and recognize DNA lesions, ultimately only flipping out the damaged substrate into the active site. A unique DNA glycosylase, the Bacillus cereus AlkD enzyme, has been shown to excise damaged DNA without flipping the nucleobase into a protein binding pocket following lesion recognition. Here, we use microsecond-scale all-atom molecular dynamics simulations to characterize the AlkD recognition mechanism, putting it in perspective with other DNA glycosylases. We first identify and describe two distinct enzyme-DNA conformations of AlkD: the search complex (SC) and excision complex (EC). The SC is distinguished by the linearity of DNA, changes in four helical parameters in the vicinity of the lesion, and changes in distance between active site residues and the DNA. Free DNA simulations are used to demonstrate that the DNA structural deviations and increased active site interactions present in the EC are initiated by the recognition of a methylation-induced signal in the rises both 5' to the methylation and opposing this base. Our results support the hypothesis that subtle geometric distortions in DNA are recognized by AlkD and are consequently probed to initiate concerted protein and DNA conformational changes which prime excise without additional intermediate states. This mechanism is shown to be consistent among the three methylated DNA sequences that have been crystallized bound to AlkD.

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.