Enhanced Sampling Molecular Dynamics Research Articles

Enhancing the Inhomogeneous Photodynamics of Canonical Bacteriophytochrome.

The ability of phytochromes to act as photoswitches in plants and microorganisms depends on interactions between a bilin-like chromophore and a host protein. The interconversion occurs between the spectrally distinct red (Pr) and far-red (Pfr) conformers. This conformational change is triggered by the photoisomerization of the chromophore D-ring pyrrole. In this study, as a representative example of a phytochrome-bilin system, we consider biliverdin IXα (BV) bound to bacteriophytochrome (BphP) from Deinococcus radiodurans. In the absence of light, we use an enhanced sampling molecular dynamics (MD) method to overcome the photoisomerization energy barrier. We find that the calculated free energy (FE) barriers between essential metastable states agree with spectroscopic results. We show that the enhanced dynamics of the BV chromophore in BphP contributes to triggering nanometer-scale conformational movements that propagate by two experimentally determined signal transduction pathways. Most importantly, we describe how the metastable states enable a thermal transition known as the dark reversion between Pfr and Pr, through a previously unknown intermediate state of Pfr. We present the heterogeneity of temperature-dependent Pfr states at the atomistic level. This work paves a way toward understanding the complete mechanism of the photoisomerization of a bilin-like chromophore in phytochromes.

PathDetect-SOM: A Neural Network Approach for the Identification of Pathways in Ligand Binding Simulations.

Understanding the process of ligand–protein recognition is important to unveil biological mechanisms and to guide drug discovery and design. Enhanced-sampling molecular dynamics is now routinely used to simulate the ligand binding process, resulting in the need for suitable tools for the analysis of large data sets of binding events. Here, we designed, implemented, and tested PathDetect-SOM, a tool based on self-organizing maps to build concise visual models of the ligand binding pathways sampled along single simulations or replicas. The tool performs a geometric clustering of the trajectories and traces the pathways over an easily interpretable 2D map and, using an approximate transition matrix, it can build a graph model of concurrent pathways. The tool was tested on three study cases representing different types of problems and simulation techniques. A clear reconstruction of the sampled pathways was derived in all cases, and useful information on the energetic features of the processes was recovered. The tool is available at https://github.com/MottaStefano/PathDetect-SOM.

CLIPSing Melanotan-II to Discover Multiple Functionally Selective hMCR Agonists.

The pleiotropic role played by melanocortin receptors (MCRs) in both physiological and pathological processes has stimulated medicinal chemists to develop synthetic agonists/antagonists with improved potency and selectivity. Here, by deploying the Chemical Linkage of Peptide onto Scaffolds strategy, we replaced the lactam cyclization of melanotan II (MT-II), a potent and unselective agonist of human MCRs (hMCRs), with different xylene-derived thioethers. The newly designed peptides displayed binding affinities toward MCRs ranging from the low nanomolar to the sub-micromolar range, highlighting a correlation between the explored linkers and the affinity toward hMCRs. In contrast to the parent peptide (MT-II), compound 5 displayed a remarkable functional selectivity toward the hMC1R. Enhanced sampling molecular dynamics simulations were found to be instrumental in outlining how the employed cyclization strategy affects the peptides' conformational behavior and, as a consequence, the detected hMC1R affinity. Additionally, a model of the peptide 5/hMC1R complex employing the very recently reported cryogenic electron microscopy receptor structure was provided.

Details of G-protein coupled receptor activation via data-driven molecular modeling

G protein-coupled receptors (GPCRs) shift between inactive non-signalling states and active signalling states, to which intracellular binding partners can bind. Extracellular binding of ligands stabilizes different receptor states and modulates the intracellular response via a complex and not well understood allosteric process. Despite the recent advances in structure determination and spectroscopy techniques, a comprehensive view of the ligand-protein interplay remains a challenge. We have derived the activation free energy landscape of the β2 adrenergic receptor (β2AR) bound to ligands with different efficacy profiles in the presence or absence of intracellular binding partner using enhanced sampling molecular dynamics (MD) simulations, to understand their modulatory effect.

Investigation into the effect of dimerization on PKR activation energetics and dynamics with enhanced sampling molecular dynamics

Protein kinase R (PKR) is a serine/threonine kinase that acts as a central component of the innate immune pathway, the cell’s first line of defense against pathogens, by phosphorylating its substrate, eIF2α, and halting translation. PKR is activated upon binding viral dsRNA, leading to autophosphorylation within the activation loop. Crystal structures and mutational scanning suggest that activation of PKR requires homodimerization in a back-to-back (BTB) geometry. To investigate, we use a computational approach to calculate the free energy changes associated with activation in the context of a monomer and a BTB dimer.

Enhanced sampling molecular dynamics to understand force response in peptide tension sensors

Mechanical forces are ubiquitous in diverse cellular processes, such as cell division, motility, and adhesion. “Mechanoresponsive” proteins and peptides are present which respond to applied forces and change in structure and function to facilitate these cellular processes. Experimental techniques such as single-force spectroscopy have been invaluable in studying protein mechanics, including force-extension curves. Scientists have also developed Tension Sensor Modules which have been used to determine the magnitude and location of forces inside cells.

Developing inhibitors of the SARS-CoV-2 main protease

SARS-CoV-2 is the causative virus of the global pandemic disease COVID-19, causing a significant public health burden. With the continued spread of SARS-CoV-2 comes an urgent need for drugs to treat those infected. The main protease (Mpro, also known as 3CL) of SARS-CoV-2 is a validated drug target. We created a pipeline to computationally screen molecules on a variety of Mpro pockets and pocket sizes, identified with enhanced sampling molecular dynamics simulations before carrying the putative hits forward to in vitro and in vivo assays for hit validation and optimization.

Peptide Dynamics and Metadynamics: Leveraging Enhanced Sampling Molecular Dynamics to Robustly Model Long-Timescale Transitions.

Molecular dynamics simulations can in theory reveal the thermodynamics and kinetics of peptide conformational transitions at atomic-level resolution. However, even with modern computing power, they are limited in the timescales they can sample, which is especially problematic for peptides that are fully or partially disordered. Here, we discuss how the enhanced sampling methods accelerated molecular dynamics (aMD) and metadynamics can be leveraged in a complementary fashion to quickly explore conformational space and then robustly quantify the underlying free energy landscape. We apply these methods to two peptides that have an intrinsically disordered nature, the histone H3 and H4 N-terminal tails, and use metadynamics to compute the free energy landscape along collective variables discerned from aMD simulations. Results show that these peptides are largely disordered, with a slight preference for α-helical structures.

Homology Modeling and Molecular Dynamics Simulations of Trypanosoma cruzi Phosphodiesterase b1.

Cyclic nucleotide phosphodiesterases have been implicated in the proliferation, differentiation and osmotic regulation of trypanosomatids; in some trypanosomatid species, they have been validated as molecular targets for the development of new therapeutic agents. Because the experimental structure of Trypanosoma cruzi PDEb1 (TcrPDEb1) has not been solved so far, an homology model of the target was created using the structure of Trypanosoma brucei PDEb1 (TbrPDEb1) as a template. The model was refined by extensive enhanced sampling molecular dynamics simulations, and representative snapshots were extracted from the trajectory by combined clustering analysis. This structural ensemble was used to develop a structure-based docking model of the target. The docking accuracy of the model was validated by redocking and cross-docking experiments using all available crystal structures of TbrPDEb1, whereas the scoring accuracy was validated through a retrospective screen, using a carefully curated dataset of compounds assayed against TbrPDEb1 and/or TcrPDEb1. Considering the results from in silico validations, the model may be applied in prospective virtual screening campaigns to identify novel hits, as well as to guide the rational design of potent and selective inhibitors targeting this enzyme.

Allosteric Effect of Nanobody Binding on Ligand-Specific Active States of the β2 Adrenergic Receptor.

Nanobody binding stabilizes G-protein-coupled receptors (GPCR) in a fully active state and modulates their affinity for bound ligands. However, the atomic-level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like β2 adrenergic receptor (β2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes structural features of the β2AR with an ∼14 Å outward movement of transmembrane helix 6 and a close proximity of transmembrane (TM) helices 5 and 7, and favors the fully active-like conformation of the receptor, independent of ligand binding, in contrast to the conditions under which no intracellular binding partner is bound, in which case the receptor is only stabilized in an intermediate-active state. This activation is supported by the residues located at hotspots located on TMs 5, 6, and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intracellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding triggers tighter and stronger local communication networks between the Nb80 and the ligand-binding sites, primarily involving residues around ICL2 and the intracellular end of TM3, TM5, TM6, as well as ECL2, ECL3, and the extracellular ends of TM6 and TM7. In particular, we identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in conformations modulated by a full agonist, BI167107, and a G-protein-biased partial agonist, salmeterol, involving mainly TM1 and TM2, and TM5, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which should be taken into account in structure-based drug discovery.

Binding free energy decomposition and multiple unbinding paths of buried ligands in a PreQ1 riboswitch.

Riboswitches are naturally occurring RNA elements that control bacterial gene expression by binding to specific small molecules. They serve as important models for RNA-small molecule recognition and have also become a novel class of targets for developing antibiotics. Here, we carried out conventional and enhanced-sampling molecular dynamics (MD) simulations, totaling 153.5 μs, to characterize the determinants of binding free energies and unbinding paths for the cognate and synthetic ligands of a PreQ1 riboswitch. Binding free energy analysis showed that two triplets of nucleotides, U6-C15-A29 and G5-G11-C16, contribute the most to the binding of the cognate ligands, by hydrogen bonding and by base stacking, respectively. Mg2+ ions are essential in stabilizing the binding pocket. For the synthetic ligands, the hydrogen-bonding contributions of the U6-C15-A29 triplet are significantly compromised, and the bound state resembles the apo state in several respects, including the disengagement of the C15-A14-A13 and A32-G33 base stacks. The bulkier synthetic ligands lead to significantly loosening of the binding pocket, including extrusion of the C15 nucleobase and a widening of the C15-C30 groove. Enhanced-sampling simulations further revealed that the cognate and synthetic ligands unbind in almost opposite directions. Our work offers new insight for designing riboswitch ligands.

Modeling the native ensemble of PhuS using enhanced sampling MD and HDX-ensemble reweighting

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

CIFDock: A novel CHARMM-based flexible receptor-flexible ligand docking protocol.

Docking studies play a critical role in the current workflow of drug discovery. However, limitations may often arise through factors including inadequate ligand sampling, a lack of protein flexibility, scoring function inadequacies (e.g., due to metals, co-factors, etc.), and difficulty in retaining explicit water molecules. Herein, we present a novel CHARMM-based induced fit docking (CIFDock) workflow that can circumvent these limitations by employing all-atom force fields coupled to enhanced sampling molecular dynamics procedures. Self-guided Langevin dynamics simulations are used to effectively sample relevant ligand conformations, side chain orientations, crystal water positions, and active site residue motion. Protein flexibility is further enhanced by dynamic sampling of side chain orientations using an expandable rotamer library. Steps in the procedure consisting of fixing individual components (e.g., the ligand) while sampling the other components (e.g., the residues in the active site of the protein) allow for the complex to adapt to conformational changes. Ultimately, all components of the complex-the protein, ligand, and waters-are sampled simultaneously and unrestrained with SGLD to capture any induced fit effects. This modular flexible docking procedure is automated using CHARMM scripting, interfaced with SLURM array processing, and parallelized to use the desired number of processors. We validated the CIFDock procedure by performing cross-docking studies using a data set comprised of 21 pharmaceutically relevant proteins. Five variants of the CHARMM-based SWISSDOCK scoring functions were created to quantify the results of the final generated poses. Results obtained were comparable to, or in some cases improved upon, commercial docking program data.

Kinetics and Mechanism of Fentanyl Dissociation from the μ-Opioid Receptor.

Driven by illicit fentanyl, opioid related deaths have reached the highest level in 2020. Currently, an opioid overdose is resuscitated by the use of naloxone, which competitively binds and antagonizes the μ-opioid receptor (mOR). Thus, knowledge of the residence times of opioids at mOR and the unbinding mechanisms is valuable for assessing the effectiveness of naloxone. In the present study, we calculate the fentanyl-mOR dissociation time and elucidate the mechanism by applying an enhanced sampling molecular dynamics (MD) technique. Two sets of metadynamics simulations with different initial structures were performed while accounting for the protonation state of the conserved H2976.52, which has been suggested to modulate the ligand-mOR affinity and binding mode. Surprisingly, with the Nδ-protonated H2976.52, fentanyl can descend as much as 10 Å below the level of the conserved D1473.32 before escaping the receptor and has a calculated residence time τ of 38 s. In contrast, with the Nϵ- and doubly protonated H2976.52, the calculated τ are 2.6 and 0.9 s, respectively. Analysis suggests that formation of the piperidine–Hid297 hydrogen bond strengthens the hydrophobic contacts with the transmembrane helix (TM) 6, allowing fentanyl to explore a deep pocket. Considering the experimental τ of ∼4 min for fentanyl and the role of TM6 in mOR activation, the deep insertion mechanism may be biologically relevant. The work paves the way for large-scale computational predictions of opioid dissociation rates to inform evaluation of strategies for opioid overdose reversal. The profound role of the histidine protonation state found here may shift the paradigm in computational studies of ligand–receptor kinetics.

Intercalation Behavior of Water-in-Salt Electrolytes in Nanoscale Hydrophobic Carbon Confinement

Intercalation of water-in-salt (WIS) electrolytes in nanoscale confinement is an important phenomenon relevant to the energy storage applications. Here, we use enhanced sampling molecular dynamics simulations to investigate the effects of intersurface separation on the structure and free energy underlying the intercalation of the Li bis(trifluoromethane)sulfonimide ([Li][TFSI]) water-in-salt (WIS) electrolyte confined between nanoscale hydrophobic carbon surfaces. We observe that the confinement separation and the WIS concentration governs the relative stability of the condensed and vapor phases of WIS inside the confinement. The relative height of the free energy barrier separating the condensed and vapor phases of WIS also strongly depends on the concentration of [Li][TFSI] inside the confined space. We find that this concentration dependence is majorly due to changes in line tension. The process of deintercalation of electrolyte passes through a vapor tube formation inside the confined space. The size of the critical vapor tube required for spontaneous evaporation of WIS from the confinement is found to be dependent on the intersurface separation and WIS concentration.

A Conserved Structural Role for the Walker-A Lysine in P-Loop Containing Kinases.

Bacterial tyrosine kinases (BY-kinases) and shikimate kinases (SKs) comprise two structurally divergent P-loop containing enzyme families that share similar catalytic site geometries, most notably with respect to their Walker-A, Walker-B, and DxD motifs. We had previously demonstrated that in BY-kinases, a specific interaction between the Walker-A and Walker-B motifs, driven by the conserved “catalytic” lysine housed on the former, leads to a conformation that is unable to efficiently coordinate Mg2+•ATP and is therefore incapable of chemistry. Here, using enhanced sampling molecular dynamics simulations, we demonstrate that structurally similar interactions between the Walker-A and Walker-B motifs, also mediated by the catalytic lysine, stabilize a state in SKs that deviates significantly from one that is necessary for the optimal coordination of Mg2+•ATP. This structural role of the Walker-A lysine is a general feature in SKs and is found to be present in members that encode a Walker-B sequence characteristic of the family (Coxiella burnetii SK), and in those that do not (Mycobacterium tuberculosis SK). Thus, the structural role of the Walker-A lysine in stabilizing an inactive state, distinct from its catalytic function, is conserved between two distantly related P-loop containing kinase families, the SKs and the BY-kinases. The universal conservation of this element, and of the key characteristics of its associated interaction partners within the Walker motifs of P-loop containing enzymes, suggests that this structural role of the Walker-A lysine is perhaps a widely deployed regulatory mechanism within this ancient family.

Conformational Properties of Glycosaminoglycan Disaccharides: A Molecular Dynamics Study.

The structure and conformation of glycosaminoglycans (GAGs) are of central importance to understand the mechanisms behind their functions in biological systems. Due to the inherent chemical and structural heterogeneity of GAGs, focusing on longer, naturally existing GAG chains hinders drawing conclusions on the influence of the chemical functionalization on the basic conformational degree of freedom, that is, the dynamic shape of glycosidic linkage present in the particular disaccharide repeating unit. In the present study, we have considered the complete set of 106 GAG-related disaccharides, being potential building blocks for longer GAG chains (including hyaluronan, chondroitin, keratan, dermatan, and heparan). Both the unfunctionalized units and all possible combinations of either partially or fully sulfated derivatives contribute to this number. The unbiased and enhanced sampling molecular dynamics simulations provide a link to understand the influence of sulfation on the conformational properties of GAG glycosidic linkages. Residue-residue hydrogen bonding is not significant for either the glycosidic linkage conformation or its flexibility. It was found that in the majority of cases, the dominating conformation of the linkage is weakly affected by sulfation and the main role is played by the steric and stereoelectronic effects. However, there exist numerous cases where sulfation increases the contribution of alternative conformations to a nonnegligible extent and, in some rare cases (restricted to disaccharides building heparan), leads to the reorientation of the glycosidic linkage. The identified sulfation sites, being the most important in this context, are C6 and C3 at the GlcNAc residue. Finally, the full set of free energy maps relying on the glycosidic dihedral angle values for diverse GAG disaccharides are provided; they may be used for further studies, focused on longer GAG chains.

The Iron Maiden. Cytosolic Aconitase/IRP1 Conformational Transition in the Regulation of Ferritin Translation and Iron Hemostasis

Maintaining iron homeostasis is fundamental for almost all living beings, and its deregulation correlates with severe and debilitating pathologies. The process is made more complicated by the omnipresence of iron and by its role as a fundamental component of a number of crucial metallo proteins. The response to modifications in the amount of the free-iron pool is performed via the inhibition of ferritin translation by sequestering consensus messenger RNA (mRNA) sequences. In turn, this is regulated by the iron-sensitive conformational equilibrium between cytosolic aconitase and IRP1, mediated by the presence of an iron–sulfur cluster. In this contribution, we analyze by full-atom molecular dynamics simulation, the factors leading to both the interaction with mRNA and the conformational transition. Furthermore, the role of the iron–sulfur cluster in driving the conformational transition is assessed by obtaining the related free energy profile via enhanced sampling molecular dynamics simulations.

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2.

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.