Haley M. Michel

Intrinsically disordered proteins (IDPs) play critical roles in cellular signaling and regulation, yet their dynamic conformational landscapes make them difficult to characterize experimentally and computationally. Phosphorylation, one of the most common post-translational modifications, frequently occurs within intrinsically disordered regions and can modulate protein structure and function. Enhanced sampling molecular dynamics methods offer a potential route to more efficiently explore the diverse conformations accessible to IDPs, but systematic comparisons of their performance sampling the conformational landscape of such systems remain limited. Here, we evaluate the conformational sampling of the intrinsically disordered β-catenin_17-48 peptide in both its nonphosphorylated and phosphorylated states using three enhanced sampling approaches: Gaussian-accelerated molecular dynamics (GaMD), metadynamics (METAD), and weighted ensemble simulations (WESTPA), in comparison with conventional molecular dynamics simulations. Two collective variables (CVs) were explored to guide sampling: the ϕ dihedral angles of the phosphorylation sites Ser33 and Ser37 and the end-to-end distance of the peptide. We found that different enhanced sampling methods explored distinct regions of conformational space rather than converging to a single ensemble, with GaMD largely overlapping with unbiased simulations. Notably, METAD and WESTPA more readily accessed conformational regions not observed in unbiased simulations. Analysis of the combined conformational ensembles identified intermediate conformations connecting the nonphosphorylated and phosphorylated states, which are preferentially sampled in simulations employing adaptive strategies. Additionally, the Ser33/Ser37 ϕ angle CV more effectively captures phosphorylation-dependent conformational shifts than the end-to-end distance metric. Together, these results highlight how both the choice of enhanced sampling strategy and the selection of collective variables influence the exploration of IDP conformational landscapes.

The growing interest in nucleic acids as therapeutic targets has prompted the devel-opment of novel computational methods to facilitate drug discovery. In this study, we extend the Site Identification by Ligand Competitive Saturation (SILCS) methodology to characterize ligand–nucleic acid interactions using the Drude polarizable force field. We demonstrate the ability of the Drude force field to better model solute-nucleic acid interactions, resulting in improved identification of known binding sites and ligand binding favorability predictions across a diverse set of nucleic acid structures. This new workflow addresses limitations in previous SILCS studies by exploiting the en-hanced sampling of solutes in the original SILCS-RNA workflow, accurately modeling the interactions of charged species, and improving solute sampling in minor groove binding sites. These results establish this Drude-based SILCS workflow as a valuable tool for structure-based drug design targeting nucleic acids and offer insights into solute preferences that can guide rational ligand design.

Gaussian-accelerated molecular dynamics (GaMD) simulations are an advanced technique that enhances the sampling of configurational space by applying biasing potentials that reduce energy barriers, enabling faster exploration of the free energy landscape. This tutorial demonstrates the application of GaMD to the alanine dipeptide, serving as an accessible model system, and guides users through all GaMD simulation stages: conventional MD, GaMD equilibration, GaMD production, and reweighting. Users will gain practical insights into the preparation of input files, monitoring of GaMD convergence, and analysis of free energy profiles using PyReweighting. We make a particular effort to connect the underlying theory with the GaMD workflow. This tutorial is intended for users with prior molecular dynamics experience, Linux and command-line navigation, and with basic Python knowledge. The step-by-step instructions and accompanying scripts aim to streamline the GaMD workflow, making it accessible for the broader research community to explore enhanced sampling for a range of biomolecular systems.

An accurate force field (FF) is the foundation of reliable results from molecular dynamics (MD) simulations. In our recently published work, we developed a protocol to generate atom pair-specific Lennard-Jones (known as NBFIX in CHARMM) and through-space Thole dipole screening (NBTHOLE) parameters in the context of the Drude polarizable FF based on readily accessible quantum mechanical (QM) data to fit condensed phase experimental thermodynamic benchmarks, including the osmotic pressure, diffusion coefficient, ionic conductivity, and solvation free energy, when available. In the present work, the developed protocol is applied to generate NBFIX and NBTHOLE parameters for interactions between monatomic ions (specifically Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, and Cl^-) and common functional groups found in proteins and nucleic acids. The parameters generated for each ion-functional group pair were then applied to the corresponding functional groups within proteins or nucleic acids followed by MD simulations to analyze the distribution of ions around these biomolecules. The modified FF successfully addresses the issue of overbinding observed in a previous iteration of the Drude FF. Quantitatively, the model accurately reproduces the effective charge of proteins and demonstrates a level of charge neutralization for a double-helix B-DNA in good agreement with the counterion condensation theory. Additionally, simulations involving ion competition correlate well with experimental results, following the trend Li⁺ > Na⁺ ≈ K⁺ > Rb⁺. These results validate the refined model for group 1 ion-biomolecule interactions that will facilitate the application of the polarizable Drude FF in systems in which group 1 ions play an important role.

G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS), can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III , is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ. SIGNIFICANCE: Here, we characterize the quadruplex:duplex junction of the HIV-1 LTR-III G-quadruplex. We find that two additional base pairs can form in the junction and are driven by electrostatic, thermodynamic, and geometric factors. G-quadruplexes containing such junctions are rather recent discoveries, and it has been proposed that these junctions can act as selective targets for drugs. These results further identify distinct chemical and electrostatic characteristics that can be used to guide drug design studies.

Telomeric DNA is guanine-rich and can adopt structures such as G-quadruplexes (GQs) and G-hairpins. Telomeric GQs influence genome stability and telomerase activity, making understanding of enzyme–GQ interactions and dynamics important for potential drug design. GQs have a characteristic tetrad core, which is connected by loop regions. Within this architecture are G-hairpins, fold-back motifs that are thought to represent the first intermediate in GQ folding. To better understand the relationship between G-hairpin motifs and GQs, we performed polarizable simulations of a two-tetrad telomeric GQ and an isolated SC11 telomeric G-hairpin. The telomeric GQ contains a G-triad, which functions as part of the tetrad core or linker regions, depending on local conformational change. This triad and another motif below the tetrad core frequently bound ions and may represent druggable sites. Further, we observed the unbiased formation of a G-triad and a G-tetrad in simulations of the SC11 G-hairpin and found that cations can be partially hydrated while facilitating the formation of these motifs. Finally, we demonstrated that K+ ions form specific interactions with guanine bases, while Na+ ions interact nonspecifically with bases in the structure. Together, these simulations provide new insights into the influence of ions on GQs, G-hairpins, and G-triad motifs.