Condensates formed by oppositely charged intrinsically disordered proteins provide model systems for understanding how transient electrostatic interactions govern structure and dynamics in biomolecular assemblies. Here we investigate a nearly charge-neutral condensate composed of 50 Prothymosin alpha (ProTalpha) and 40 Histone H1 molecules using a single-bead-per-residue coarse-grained model combining the HPS hydropathy model for disordered regions with a Go model for the globular domain of Histone H1 under NPT conditions at pressures from 2 to 12 bar. We find that chain dimensions, including the radius of gyration (Rg), end-to-end distance (Ree), and their ratio R, are insensitive to pressure, indicating that chain conformations remain largely unchanged over the pressure range studied. Histone H1 exhibits systematically larger values of R than ProTalpha because of its globular-core plus disordered-tail architecture. Translational diffusion coefficients decrease monotonically with pressure, from approximately 0.22 to 0.06 nm^2/ns, with substantial chain-to-chain heterogeneity comparable to the mean diffusion coefficient. Chain relaxation follows a stretched exponential with beta less than 1 that decreases with pressure. ProTalpha relaxation times of approximately 12 to 40 ns obey Rouse scaling, whereas Histone H1 deviates because of the internal constraint imposed by its globular domain. ProTalpha-Histone H1 contact lifetimes of approximately 0.43 to 0.56 ns are much shorter than the Rouse relaxation time, placing the system firmly in the fast-exchange regime where transient electrostatic contacts renormalize chain friction rather than acting as permanent cross-links, consistent with the moderate stretching exponent beta of approximately 0.55 to 0.70 observed across all pressures.
Leucine zippers are parallel coiled-coil dimers composed of two α-helices with a repeating heptad pattern, designated as a–g. Hydrophobic residues at the a and d positions form the dimerization core, whereas charged residues at the e and g positions generate interhelical electrostatic interactions that influence stability and specificity. Oppositely charged e–g pairs are generally considered stabilizing, whereas like-charged pairs are expected to be destabilizing. However, their collective effects across an extended leucine zipper interface have not been systematically evaluated. Here, we designed a 40-residue homodimeric leucine zipper containing 10 e–g interaction positions. Ten e–g positions were independently assigned to one of three states—neutral, salt-bridge-forming, or repulsive—yielding 59,049 models. Each model was then analyzed with FoldX to calculate the interaction energy and component energy terms. Increasing the number of salt bridges progressively improved the interaction energy, with an average stabilization of 1.68 kcal/mol relative to neutral pairs. Unexpectedly, repulsive pairs were also modestly favorable relative to neutral pairs, improving interaction energy by 0.48 kcal/mol. Energy decomposition showed that favorable solvation and van der Waals contributions outweighed the electrostatic penalty. These effects were position-dependent, indicating that leucine zipper stability reflects a balance of electrostatics, solvation, packing, and positional context.
Ring-like AAA+ (ATPases Associated with diverse cellular Activities) biological machines mediate protein remodeling to assist a broad range of essential cellular functions. The nucleotide-dependent remodeling action involves intra- and inter-ring allosteric communication to generate mechanical force applied onto the substrate by a set of loops that protrude into the central channel. In this study, we probe these allosteric mechanisms through a comparative study of the katanin, a microtubule severing protein including a clade 3 AAA domain, and the double-ring ClpB, a protein disaggregase including both a clade 3 and a clade 5 AAA domain. Our molecular dynamics simulations, combined with machine learning and bioinformatic analysis, reveal both similar mechanisms involving the clade 3 domain and ClpB-specific ones involving communication with the clade 5 domain. We find that both nucleotide and substrate polypeptide binding restrict the conformational landscape sampled by katanin and ClpB, with ligand-specific conformations observed in the latter case. Allosteric contributions of secondary structure elements, ranked by using SHapley Additive exPlanations analysis in machine learning approaches and binary classification of features in ligand states, highlight the important role of regions adjacent to the nucleotide-binding site and the pore loops. Amino acid-level analysis of the allosteric paths reveals that intra-ring cooperativity modulates long-distance communication within the AAA+ protomers.
Many functional RNA motifs adopt structures that deviate from the canonical A-form helix and are emerging targets for RNA-directed therapeutics. The microtubule-associated protein tau (MAPT) A-bulge motif (5′-GCAGU/5′-ACGU) is one such motif. Because its structure is stabilized by a delicate balance of local interactions, its accurate modeling remains a major challenge for molecular dynamics (MD) simulations. The experimentally determined nuclear magnetic resonance (NMR) structure of the MAPT A-bulge motif provides a stringent test of whether RNA force fields can accurately reproduce the experimentally observed conformation. Most current AMBER-family RNA force-field models have incorrectly favored a non-native base-triple state of the MAPT A-bulge motif over the experimentally observed stacked state. Structural comparison of the stacked and base-triple conformations revealed that overly favorable NH□–N hydrogen bonds between the bulged adenosine and an adjacent Watson–Crick base pair were the primary source of this imbalance. We developed gHBfix-18Ab, an 18-component hydrogen-bond correction that distinguishes NH and NH□ donors. gHBfix-18Ab was combined with the previously developed OL3CP and NBfix0BPh corrections to generate the composite model gHBfix-18Ab*. This model restored the experimentally observed stacked state as the global minimum in the calculated free-energy profile and improved agreement with NMR-derived distance data for the A-bulge region. Importantly, gHBfix-18Ab* did not produce marked structural destabilization of the cUUCGg tetraloop, a widely used benchmark for RNA force-field validation, suggesting that the refinement preserves the stability of the unrelated RNA motif. These results demonstrate that targeted refinement of hydrogen-bond interactions provides a practical strategy for systematic improvement of RNA force fields toward more accurate modeling of noncanonical RNA motifs. Graphical Summary
A conserved sequence-ensemble-dynamics code in Nhp6A is revealed wherein not just stability, but also phosphorylation-induced conformational switching, disordered tail dynamics, and DNA binding-bending closely coordinate chromatin accessibility is revealed.
Shilpi Laha, H. Madhan, Yuji Itoh et al.· bioRxiv· 0 citations
Collagen's triple helix structure is fundamental to the mechanical integrity of bodily tissues, and its stability is vital for optimal physiological function. While the stabilization mechanisms of simple, symmetric collagen homotrimers have been previously studied, the hydration dynamics of native-like collagen heterotrimers which comprise the vast majority of human collagens remain largely unexplored. In this study, we investigated the complex role of water in stabilizing heterotrimeric collagen model peptides (CMPs) using extensive molecular dynamics (MD) simulations. We examined structurally diverse AAB-type and ABC-type heterotrimers exhibiting varying experimental stability profiles to investigate how sequence asymmetry influences hydration organization and collagen stability. Our simulations revealed that water molecules dynamically organize around these complex structures, forming topological water networks (TWNs) via intermolecular hydrogen bonds. Quantitative analyses of peptide-water interactions, hydration-water dynamics, and normalized TWN counts, together with residue- and chain-resolved TWN characterization, demonstrated that hydration-shell organization is strongly sequence dependent and differs among heterotrimers with distinct thermal stabilities. In particular, the more thermally stable heterotrimers exhibited a greater propensity for hydration-shell water molecules to participate in cyclic TWNs, accompanied by reduced hydration-water mobility and favorable interchain hydrogen-bonding interactions. These findings extend our previous observations on collagen homotrimers and provide new atomistic insight into the sequence-dependent organization of hydration water surrounding collagen heterotrimers, suggesting that organized hydration water contributes to collagen stability in concert with interchain hydrogen bonding and hydration-water dynamics rather than acting as an independent stabilization mechanism.
Anand Balupuri, Dong-Hyun Son, Nam Sook Kang· Biopolymers· 0 citations
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