Conformational Dynamics Research Articles

Membrane curvature sensing and symmetry breaking of the M2 proton channel from Influenza A.

The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.

A Single Interfacial Point Mutation Rescues Solution Structure Determination of the Complex of HMG-D with a DNA Bulge.

Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.

Exploring Mutation-Driven Changes in the ATP-ADP Conformational Cycle of Human Hsp70 by All-Atom MD Adaptive Sampling.

Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.

Exploring the Ligand Binding and Conformational Dynamics of the Substrate-Binding Domain 1 of the ABC Transporter GlnPQ.

The adenosine triphosphate (ATP)-binding cassette (ABC) importer GlnPQ from Lactococcus lactis has two sequential covalently linked substrate-binding domains (SBDs), which capture the substrates and deliver them to the translocon. The two SBDs differ in their ligand specificities, binding affinities and the distance to the transmembrane domain; interestingly, both SBDs can bind their ligands simultaneously without affecting each other. In this work, we studied the binding of ligands to both SBDs using X-ray crystallography and molecular dynamics simulations. We report three high-resolution structures of SBD1, namely, the wild-type SBD1 with bound asparagine or arginine, and E184D SBD1 with glutamine bound. Molecular dynamics (MD) simulations provide a detailed insight into the dynamics associated with open-closed transitions of the SBDs.

Connecting Protein Millisecond Conformational Dynamics to Protein Thermal Stability.

The stability of protein folded states is crucial for its function, yet the relationship with the protein sequence remains poorly understood. Prior studies have focused on the amino acid composition and thermodynamic couplings within a single folded conformation, overlooking the potential contribution of protein dynamics. Here, we address this gap by systematically analyzing the impact of alanine mutations in the C-terminal β-strand (β5) of ubiquitin, a model protein exhibiting millisecond timescale interconversion between two conformational states differing in the β5 position. Our findings unveil a negative correlation between millisecond dynamics and thermal stability, with alanine substitutions at seemingly flexible C-terminal residues significantly enhancing thermostability. Integrating spectroscopic and computational approaches, we demonstrate that the thermally unfolded state retains a substantial secondary structure but lacks β5 engagement, recapitulating the transition state for millisecond dynamics. Thus, alanine mutations that modulate the stabilities of the folded states with respect to the partially unfolded state impact both the dynamics and stability. Our findings underscore the importance of conformational dynamics with implications for protein engineering and design.

Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death

Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Chemical Synthesis and Structure-Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata.

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure-activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.

In Silico Analysis of Conformational Dynamics and Energetic Landscapes of Putative Insulinase PF11_0189 From the Plasmodium falciparum Genome With Insulin‐Derived Peptides: Approach to Rationale Design of Insulin Peptide‐Based Inhibitors

ABSTRACTGlucose abnormality is one of the atypical symptoms mostly overlooked during severe malaria. Screening for proteases involved in glucose abnormality suggests a potential new drug target. PF11_0189 is a putative insulin degrading enzyme found in the genome of Plasmodium falciparum. In silico approach revealed that PF11_0189 is similar to the metalloproteases exhibiting a zinc binding motif. The substrate binding region of PF11_0189 catalytic domain is lined by residues of a mixed nature, enabling the accommodation of both hydrophobic and hydrophilic residues from substrates. Analysis of each amino acid position from combinatorial peptide library suggests Thr and Ser to be the most crucial residues as upon its mutation significant improvement is observed in the binding. The peptides P‐21 (VPICSLY), P‐70 (TMICVLY), and P‐121 (AVICSLY) demonstrate significantly better interaction within the active site than a template peptide (TSICSLY). Molecular dynamic simulations confirm the complex's integrity, with all structures within the qualitative limit of compactness and stability during the simulation time. Principal component analysis (PCA) reveals major conformational motions and different energy landscapes, while dynamic cross‐correlation matrix (DCCM) highlights residue interactions of the complexes. The insights obtained through computational methods provide clues about substrate selectivity in PF11_0189, which can be utilized for peptide‐based therapeutics against severe malaria.

Junctions at the crossroads: the impact of mechanical cues on endothelial cell-cell junction conformations and vascular permeability.

Cells depend on precisely regulating barrier function within the vasculature to maintain physiological stability and facilitate essential substance transport. Endothelial cells achieve this through specialized adherens and tight junction protein complexes, which govern paracellular permeability across vascular beds. Adherens junctions, anchored by vascular endothelial (VE)-cadherin and associated catenins to the actin cytoskeleton, mediate homophilic adhesion crucial for barrier integrity. In contrast, tight junctions composed of occludin, claudin, and junctional adhesion molecule A interact with Zonula Occludens proteins, reinforcing intercellular connections essential for barrier selectivity. Endothelial cell-cell junctions exhibit dynamic conformations during development, maturation, and remodeling, regulated by local biochemical and mechanical cues. These structural adaptations play pivotal roles in disease contexts such as chronic inflammation, where junctional remodeling contributes to increased vascular permeability observed in conditions from cancer to cardiovascular diseases. Conversely, the brain microvasculature's specialized junctional arrangements pose challenges for therapeutic drug delivery due to their unique molecular compositions and tight organization. This commentary explores the molecular mechanisms underlying endothelial cell-cell junction conformations and their implications for vascular permeability. By highlighting recent advances in quantifying junctional changes and understanding mechanotransduction pathways, we elucidate how physical forces from cellular contacts and hemodynamic flow influence junctional dynamics.

Dependence of photophysical properties on stereoisomers: The case of nonsymmetric 2-aryl(benz)imidazole-annulated derivatives

Nonsymmetric 2-aryl (benz)imidazole derivatives were previously synthesized via C-H/N-H annulation of imidazole derivatives from lapachol moieties with internal alkynes as an efficient tool to increase the π-system of lapimidazole derivatives. To investigate their photophysical properties, time-resolved and steady-state fluorescence measurements were carried out on dropcast film, powder, and diluted solution. Despite having the same number of single and double bonds, slight differences in their structures lead to distinct photophysical properties. The time-correlated single-photon counting measurements also demonstrated that the aspects of the temporal decay curves are dependent on the treatment of the samples during the measurements. The decay curves were relatively well fitted using an alternative method based on the exponentially modified Gaussian function, giving rise to a more complete set of fitting parameters. Finally, theoretical calculations corroborate that small structural changes in molecules can significantly alter their conformational dynamics, electronic spectra, and overall electronic behavior and stability.

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding.

Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.

Integration of AlphaFold with Molecular Dynamics for Efficient Conformational Sampling of Transporter Protein NarK.

Transporter proteins carry their substrate across the cell membrane by changing their conformation. Thus, conformational dynamics are crucial for transport function. However, clarifying the complete transport cycle is challenging even with the current structural biology approach. Molecular dynamics (MD) simulation is a computational approach that can provide the time-resolved conformational dynamics of transporter proteins in atomic details but suffers from a high computational cost. Here, we integrate state-of-the-art protein structure prediction AI, AlphaFold2 (AF2), with MD simulation to reduce the computational cost. Focusing on the transporter protein NarK, we first show that AF2 sampled broad conformations of NarK, including the inward-open, occluded, and outward-open states. We also applied the coevolution-informed mutation in AF2, identifying state-shifting mutations. Then, we show that MD simulations from AF2-generated outward-open conformation, which is experimentally unresolved, captured the essence of the conformational state. We also found that MD simulations from AF2-generated intermediates showed transient dynamics like a transition state connecting two conformational states. This study paves the way for efficient conformational sampling of transporter proteins.

Molecular Dynamics Simulations of the Eye Lens Water Channel Aquaporin 0 from Fish.

Aquaporin 0 (AQP0) plays a key role in water circulation in the eye lens through a variety of functions. In contrast to mammalian genomes, zebrafish contains two aqp0 genes leading to a separation of AQP0 multiple functions between the two gene products, Aqp0a and Aqp0b. A notable feature of the zebrafish AQP0 paralogs is the increased water permeability of Aqp0b relative to Aqp0a as well as a severa lfold increase relative to mammalian AQP0. Here, we report equilibrium molecular dynamics (MD) simulations on the microsecond timescale to identify the structural basis underlying the differences in water permeability between zebrafish AQP0 paralogs and between AQP0 mammalian and fish orthologs. Our simulations are able to reproduce the experimental trends in water permeability. Our results suggest that a substitution of a key Y23 residue in mammalian AQP0 for F23 in fish AQP0 orthologs introduces significant changes in the conformational dynamics of the CS-I structural motif, which, in conjunction with different levels of hydration of the channel vestibule, can account for the differences in permeabilities between fish and mammalian AQP0 orthologs and between zebrafish AQP0 paralogs.

The interaction of the azetidine thiazole side chain with the active site loop (ASL) 3 drives the evolution of IMP metallo-β-lactamase against tebipenem.

Imipenemase (IMP) metallo-β-lactamases (MBLs) hydrolyze almost all available β-lactams including carbapenems and are not inhibited by any commercially available β-lactamase inhibitor. Tebipenem (TP) pivoxil is the first orally available carbapenem and possesses a unique bicyclic azetidine thiazole moiety located at the R2 position. TP has potent in vitro activity against Enterobacterales producing extended-spectrum and/or AmpC β-lactamases. Thus far, the activity of TP against IMP-producing strains is understudied. To address this knowledge gap, we explored the structure activity relationships of IMP MBLs by investigating whether IMP-6, IMP-10, IMP-25, and IMP-78 [MBLs with expanded hydrolytic activity against meropenem (MEM)] would demonstrate enhanced activity against TP. Most of the Escherichia coli DH10B strains expressing IMP-1 variants displayed a ≥twofold MIC difference between TP and MEM, while those expressing VIM or NDM variants demonstrated comparable MICs. Catalytic efficiency (kcat/KM) values for the TP hydrolysis by IMP-1, IMP-6, IMP-10, IMP-25, and IMP-78 were significantly lower than those obtained for MEM. Molecular dynamic simulations reveal that V67F and S262G substitutions (found in IMP-78) reposition active site loop 3, ASL-3, to better accommodate the bicyclic azetidine thiazole side chain, allowing microbiological/catalytic activity to approach that of comparison MBLs used in this study. These findings suggest that modifying the R2 side chain of carbapenems can significantly impact hydrolytic stability. Furthermore, changes in conformational dynamics due to single amino acid substitutions should be used to inform drug design of novel carbapenems.

Molecular Insights into the Binding and Conformational Changes of Hepcidin25 Blood Peptide with 4-Aminoantipyrine and Their Sorption Mechanism by Carboxylic-Functionalized Multiwalled Carbon Nanotubes: A Comprehensive Spectral Analysis and Molecular Dynamics Simulation Study.

In this work, the main purpose is to analyze and understand the mechanism and thermodynamic interactions of carboxylic acid-functionalized multiwalled carbon nanotubes (cf-MWCNTs) and 4-aminoantipyrine (AAP) with human hepcidine25 (Hep25) using multispectroscopic and molecular docking modeling methods, binding free energy calculations, and molecular dynamics (MD) simulations under physiological conditions. AAP belongs to a class of persistent environmental contaminants, and its residue is a potential hazard to human health, exhibiting a high binding affinity with blood peptides. Hepcidin is a 25-residue peptide hormone with four disulfide bonds that regulates the iron balance in vertebrates and contributes to host immunity as a cysteine-rich antimicrobial peptide. Due to their diverse properties and pollutant absorption capabilities, CNTs demonstrate important biological effects in biological applications, particularly in the noncovalent interactions with blood peptides. A comprehensive molecular dynamics simulation integrated with molecular docking methodologies was employed to explore the binding free energy between AAP and Hep25, identify binding sites, elucidate thermodynamic characteristics, and evaluate the binding forces governing their interaction. The investigation delved into elucidating the precise binding site of AAP within the Hep25 protein and thoroughly analyzed the impact of AAP on the microenvironment and conformational dynamics of Hep25. The circular dichroism (CD) experimental results highlight a reduction in β-sheet composition following the introduction of AAP and cf-MWCNT. In addition, outcomes from fluorescence spectroscopy demonstrate that both cf-MWCNT and AAP significantly attenuated Hep-25 fluorescence via a static quenching mechanism. According to the MD simulations, the presence of AAP induces changes in the secondary structure of Hep25 and enhances its hydrophobicity. Additionally, our findings demonstrated that alongside the alteration in protein structure and functionality induced by contaminants, cf-MWCNTs possess the capability to mitigate the contaminant-induced effects on Hep25 activity while preserving the overarching structural integrity of Hep25. Based on the distance and RDF data, we found that during the simulation the presence of the cf-MWCNT causes the AAP to move away from the Hep25, and as a result fewer and weaker interactions of the AAP with the Hep25 will be observed. Likewise, free energy calculations indicate that the binding of Hep25 to AAP and cf-MWCNT involves electrostatic, π-cationic, and π-π stacking interactions. The research findings offer invaluable insights into the intricate influence of pollutants and carbon nanotubes on protein functionality within the circulatory system and their toxicity in vivo for prospective investigations.

Unraveling the Structure-Spectrum Relationship of Yeast Phenylalanine Transfer RNA: Insights from Theoretical Modeling of Infrared Spectroscopy.

Yeast phenylalanine tRNA (tRNAphe) is a paradigmatic model in structural biology. In this work, we combine molecular dynamics simulations and spectroscopy modeling to establish a direct link between its structure, conformational dynamics, and infrared (IR) spectra. Employing recently developed vibrational frequency maps and coupling models, we apply a mixed quantum/classical treatment of the line shape theory to simulate the IR spectra of tRNAphe in the 1600-1800 cm-1 region across its folded and unfolded conformations and under varying concentrations of Mg2+ ions. The predicted IR spectra of folded and unfolded tRNAphe are in good agreement with experimental measurements, validating our theoretical framework. We then elucidate how the characteristic L-shaped tertiary structure of the tRNA and its modulation in response to diverse chemical environments give rise to distinct IR absorption peaks and line shapes. These calculations effectively bridge IR spectroscopy experiments and atomistic molecular simulations, unraveling the molecular origins of the observed IR spectra of tRNAphe. This work presents a robust theoretical protocol for modeling the IR spectroscopy of nucleic acids, which will facilitate its application as a sensitive probe for detecting the fluctuating secondary and tertiary structures of these essential biological macromolecules.

In DFT We Trust: Exhaustive Exploration of 1,3‐Dipolar Cycloadditions Between Nitrones and Levoglucosenone Exposes a Curious Case of Conformational Dynamics

AbstractAn experimental and computational study (including DFT calculations and distortion/interaction analysis) was conducted to assess the effect of the nitrone structure in the outcome of dipolar 1,3‐cycloadditions with levoglucosenone, a biomass derived chiral enone. While B3LYP/6‐31G* (the most popular method for modeling these reactions according to our literature search) provides qualitatively good results, large outliers were found for some systems versus experimental data. An exhaustive exploration of other levels allowed us to determine the most appropriate ones to predict simultaneously reactivity and selectivity. The systematically predicted high exo selectivity by the majority of the levels led us to reconsider our initial assignment for the reaction with the nitrone derived from piperidine, which resulted in the discovery of an interesting case of conformational dynamics.

Structural Snapshots of Proteus vulgaris Tryptophan Indole-Lyase Reveal Insights into the Catalytic Mechanism.

Tryptophan indole lyase (TIL; [E.C. 4.1.99.1]) is a bacterial pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes reversible β-elimination of indole from L-tryptophan. The mechanism of elimination of indole from L-tryptophan starts with the formation of an external aldimine of the substrate and PLP, followed by deprotonation of the α-CH of the substrate, forming a resonance-stabilized quinonoid intermediate. Proton transfer to C3 of the indole ring and carbon-carbon bond cleavage of the quinonoid intermediate provide indole and aminoacrylate bound to PLP, which then releases indole, followed by iminopyruvate. We have now determined the X-ray crystal structures of TIL complexes with (3S)-dioxindolyl-l-alanine, an inhibitor, and with substrates L-tryptophan, 7-aza-L-tryptophan, and S-ethyl-l-cysteine (SEC) in the presence of benzimidazole (BZI), an isostere of the product indole. These structures show a mixture of gem-diamine, external aldimine, quinonoid, and aminoacrylate intermediates, in both open and closed active site conformations. In the closed conformations of L-tryptophan, (3S)-dioxindolyl-l-alanine, and 7-aza-L-tryptophan complexes, hydrogen bonds form between Asp-133 with N1 of the ligand heterocyclic ring and NE2 of His-458 in the small domain of TIL. This hydrogen bond also forms in the BZI complex with the aminoacrylate intermediates formed from both L-tryptophan and SEC. The closed quinonoid complex of 7-aza-L-tryptophan shows that the azaindole ring in the closed conformation is bent out of plane of the Cβ-C3 bond by about 40°, putting it in a geometry that leads toward the transition-state geometry. Thus, both conformational dynamics and substrate activation play critical roles in the reaction mechanism of the TIL.

The Effect of N52R Mutation at the SPN-ARR Interface on the Conformational Dynamics of SHANK3

Background:: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition. The genetic basis of ASD involves numerous loci converging on neural pathways, particularly affecting excitatory synapses. SHANK3, an essential protein in the post-synaptic neurons, has been implicated in ASD, with mutations affecting its N-terminal, including the SPN domain. Objective: This study aims to investigate the impact of the N52R mutation on SHANK3 and assess the dynamics, stability, flexibility, and compactness of the N52R mutant compared to SHANK3 WT. Methods: Molecular dynamics simulations were conducted to investigate the structural dynamics of SHANK3 WT and the N52R mutant. The simulations involved heating dynamics, density equilibrium, and production dynamics. The trajectories were analyzed for RMSD, RMSF, Rg, hydrogen bond analysis, and secondary structure. Results: The simulations revealed that the N52R mutant disrupts the stability and folding of SHANK3, affecting intramolecular contacts between SPN and ARR. This disruption opens up the distance between SPN and ARR domains, potentially influencing the protein's interactions with partners, including αCaMKII and α-Fodrin. The altered conformation of the SPN-ARR tandem in the N52R mutant suggests a potential impact on dendritic spine shape and synaptic plasticity. Conclusion: The findings shed light on the structural consequences of the N52R mutation in SHANK3, emphasizing its role in influencing intramolecular interactions and potential effects on synaptic function. Understanding these molecular dynamics contributes to unraveling the intricate relationship between genetic variations in SHANK3 and clinical traits associated with ASD. Further investigations are warranted to explore the physiological implications of these structural alterations in vivo.

Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance

DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. We determined cryoelectron microscopy (cryo-EM) structures of DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antivirals in clinical use. These structures reveal how the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis. Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sampled by the fingers domain. Drug-bound structures reveal how antivirals may selectively bind enzymes that more readily adopt the closed conformation. Molecular dynamics simulations and the cryo-EM structure of a drug-resistant mutant indicate that some resistance mutations modulate conformational dynamics rather than directly impacting drug binding, thus clarifying mechanisms that drive drug selectivity.