Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

Our sensory adaptation to cold and chemically induced coolness is mediated by the intrinsic property of TRPM8 channels to desensitize. TRPM8 is also implicated in cold-evoked pain disorders and migraine, highlighting its inhibitors as an avenue for pain relief. Despite the importance, the mechanisms of TRPM8 desensitization and inhibition remained unclear. We found, using cryo-electron microscopy, electrophysiology, and molecular dynamics simulations, that TRPM8 inhibitors bind selectively to the desensitized state of the channel. These inhibitors were used to reveal the overlapping mechanisms of desensitization and inhibition and that cold and cooling agonists share a common desensitization pathway. Furthermore, we identified the structural determinants crucial for the conformational change in TRPM8 desensitization. Our study illustrates how receptor-level conformational changes alter cold sensation, providing insights into therapeutic development.

Ligand Binding Affinity Prediction for Membrane Proteins with Alchemical Free Energy Calculation Methods.

Alchemical relative binding free energy (ΔΔG) calculations have shown high accuracy in predicting ligand binding affinity and have been used as important tools in computer-aided drug discovery and design. However, there has been limited research on the application of ΔΔG methods to membrane proteins despite the fact that these proteins represent a significant proportion of drug targets, play crucial roles in biological processes, and are implicated in numerous diseases. In this study, to predict the binding affinity of ligands to G protein-coupled receptors (GPCRs), we employed two ΔΔG calculation methods: thermodynamic integration (TI) with AMBER and the alchemical transfer method (AToM) with OpenMM. We calculated ΔΔG values for 53 transformations involving four class A GPCRs and evaluated the performance of AMBER-TI and AToM-OpenMM. In addition, we conducted tests using different numbers of windows and varying simulation times to achieve reliable ΔΔG results and to optimize resource utilization. Overall, both AMBER-TI and AToM-OpenMM show good agreement with the experimental data. Our results validate the applicability of AMBER-TI and AToM-OpenMM for optimization of lead compounds targeting membrane proteins.

Binary bilayer simulations for partitioning within membranes.

Membrane proteins (MPs) often show preference for one phase over the other, which is characterized by the partition coefficient, Kp. The physical mechanisms underlying Kp have been only inferred indirectly from experiments due to the unavailability of detailed structures and compositions of ordered phases. Molecular dynamics (MD) simulations can complement these details and thus, in principle, provide further insights into the partitioning of MPs between two phases. However, the application of MD has remained difficult due to long time scales required for equilibration and large system size for the phase stability, which have not been fully resolved even in free energy simulations. This chapter describes the recently developed binary bilayer simulation method, where the membrane is composed of two laterally attached membrane patches. The binary bilayer system (BBS) is designed to preserve the lateral packing of both phases in a significantly smaller size compared to that required for macroscopic phase separation. These characteristics are advantageous in partitioning simulations, as the length scale for diffusion across the system can be significantly smaller. Hence the BBS can be efficiently employed in both conventional MD and free energy simulations, though sampling in ordered phases remains difficult due to slow diffusion. Development of efficient lipid swapping methods and its combination with the BBS would be a useful approach for partitioning in coexisting phases.

Lipid-coated gold nanorods for photoimmunotherapy of primary breast cancer and the prevention of metastasis.

Nanomedicines hold promise for the treatment of various diseases. However, treating cancer metastasis remains highly challenging. In this study, we synthesized gold nanorods (AuNRs) containing (α-GC), an immune stimulator, for the treatment of primary cancer, metastasis, and recurrence of the cancer. Therefore, the AuNR were coated with lipid bilayers loaded with α-GC (α-LA). Upon irradiation with 808 nm light, α-LA showed a temperature increase. Intra-tumoral injection of α-LA in mice and local irradiation of the 4T1 breast cancer tumor effectively eliminated tumor growth. We found that the presence of α-GC in α-LA activated dendritic cells and T cells in the spleen, which completely blocked the development of lung metastasis. In mice injected with α-LA for primary breast cancer treatment, we observed antigen-specific T cell responses and increased cytotoxicity against 4T1 cells. We conclude that α-LA is promising for the treatment of both primary breast cancer and its metastasis.

CHARMM-GUI QM/MM Interfacer for a Quantum Mechanical and Molecular Mechanical (QM/MM) Simulation Setup: 1. Semiempirical Methods.

Quantum mechanical (QM) treatments, when combined with molecular mechanical (MM) force fields, can effectively handle enzyme-catalyzed reactions without significantly increasing the computational cost. In this context, we present CHARMM-GUI QM/MM Interfacer, a web-based cyberinfrastructure designed to streamline the preparation of various QM/MM simulation inputs with ligand modification. The development of QM/MM Interfacer has been achieved through integration with existing CHARMM-GUI modules, such as PDB Reader and Manipulator, Solution Builder, and Membrane Builder. In addition, new functionalities have been developed to facilitate the one-stop preparation of QM/MM systems and enable interactive and intuitive ligand modifications and QM atom selections. QM/MM Interfacer offers support for a range of semiempirical QM methods, including AM1(+/d), PM3(+/PDDG), MNDO(+/d, +/PDDG), PM6, RM1, and SCC-DFTB, tailored for both AMBER and CHARMM. A nontrivial setup related to ligand modification, link-atom insertion, and charge distribution is automatized through intuitive user interfaces. To illustrate the robustness of QM/MM Interfacer, we conducted QM/MM simulations of three enzyme-substrate systems: dihydrofolate reductase, insulin receptor kinase, and oligosaccharyltransferase. In addition, we have created three tutorial videos about building these systems, which can be found at https://www.charmm-gui.org/demo/qmi. QM/MM Interfacer is expected to be a valuable and accessible web-based tool that simplifies and accelerates the setup process for hybrid QM/MM simulations.

CHARMM-GUI Multicomponent Assembler for modeling and simulation of complex multicomponent systems.

Atomic-scale molecular modeling and simulation are powerful tools for computational biology. However, constructing models with large, densely packed molecules, non-water solvents, or with combinations of multiple biomembranes, polymers, and nanomaterials remains challenging and requires significant time and expertise. Furthermore, existing tools do not support such assemblies under the periodic boundary conditions (PBC) necessary for molecular simulation. Here, we describe Multicomponent Assembler in CHARMM-GUI that automates complex molecular assembly and simulation input preparation under the PBC. In this work, we demonstrate its versatility by preparing 6 challenging systems with varying density of large components: (1) solvated proteins, (2) solvated proteins with a pre-equilibrated membrane, (3) solvated proteins with a sheet-like nanomaterial, (4) solvated proteins with a sheet-like polymer, (5) a mixed membrane-nanomaterial system, and (6) a sheet-like polymer with gaseous solvent. Multicomponent Assembler is expected to be a unique cyberinfrastructure to study complex interactions between small molecules, biomacromolecules, polymers, and nanomaterials.

Neuroprotective amyloid ß N-terminal peptides differentially alter human α7- and α7ß2-nicotinic acetylcholine (nACh) receptor single-channel properties.

BACKGROUND AND PURPOSE: Oligomeric amyloid ß 1-42 (oAß1-42) exhibits agonist-like action at human α7- and α7ß2-containing nicotinic receptors. The N-terminal amyloid ß1-15 fragment (N-Aß fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aß fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aßcore), protect against oAß1-42-associated synapto- and neurotoxicity. Here, we investigated how oAß1-42, the N-Aß fragment, and the N-Aßcore regulate the single-channel properties of α7- and α7ß2-nicotinic receptors. EXPERIMENTAL APPROACH: Single-channel recordings measured the impact of acetylcholine, oAß1-42, the N-Aß fragment, and the N-Aßcore on the unitary properties of human α7- and α7ß2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aß fragment and orthosteric α7+/α7- and α7+/ß2- nicotinic receptor binding interfaces. KEY RESULTS: The N-Aß fragment and N-Aßcore induced α7- and α7ß2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAß1-42 preferentially enhanced α7ß2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aßcore neutralized these effects. Further, administration of the N-Aß fragment alone, or in combination with acetylcholine or oAß1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAß1-42). CONCLUSIONS AND IMPLICATIONS: Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7ß2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7ß2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid ß-derived fragments against the toxic effects of oAß1-42.

CHARMM-GUI Membrane Builder for Lipid Droplet Modeling and Simulation.

Lipid droplets (LDs) are organelles that are necessary for eukaryotic and prokaryotic metabolism and energy storage. They have a unique structure consisting of a spherical phospholipid monolayer encasing neutral lipids such as triacylglycerol (TAG). LDs have garnered increased interest for their implications in disease and for drug delivery applications. Consequently, there is an increased need for tools to study their structure, composition, and dynamics in biological contexts. In this work, we utilize CHARMM-GUI Membrane Builder to simulate and analyze LDs with and without a plant LD protein, oleosin. The results show that Membrane Builder can generate biologically relevant all-atom LD systems with relatively short equilibration times using a new TAG library having optimized headgroup parameters. TAG molecules originally inserted into a lipid bilayer aggregate in the membrane center, forming a TAG-only core flanked by two monolayers. The TAG-only core thickness stably grows with increasing TAG mole fraction. A 70 % TAG system has a core that is thick enough to house oleosin without its interactions with the distal leaflet or disruption of its secondary structure. We hope that Membrane Builder can aid in the future study of LD systems, including their structure and dynamics with and without proteins.

CHARMM GUI Membrane Builder for oxidized phospholipid membrane modeling and simulation.

Oxidative stress leads to the production of oxidized phospholipids (oxPLs) that modulate the biophysical properties of phospholipid monolayers and bilayers. As many immune cells are responsible for surveilling cells and tissues for the presence of oxPLs, oxPL-dependent mechanisms have been suggested as targets for treating chronic kidney disease, atherosclerosis, diabetes, and cancer metastasis. This review details recent experimental and computational studies that characterize oxPLs' behaviors in various monolayers and bilayers. These studies investigate how the tail length and polar functional groups of OxPLs impact membrane properties, how oxidized membranes can be stabilized, and how membrane integrity is generally affected by oxidized lipids. In addition, for oxPL-containing membrane modeling and simulation, CHARMM-GUI Membrane Builder has been extended to support a variety of oxPLs, accelerating the simulation system building process for these biologically relevant lipid bilayers.

Type Label Framework for Bonded Force Fields in LAMMPS.

New functionality is added to the LAMMPS molecular simulation package, which increases the versatility with which LAMMPS can interface with supporting software and manipulate information associated with bonded force fields. We introduce the "type label" framework that allows atom types and their higher-order interactions (bonds, angles, dihedrals, and impropers) to be represented in terms of the standard atom type strings of a bonded force field. Type labels increase the human readability of input files, enable bonded force fields to be supported by the OpenKIM repository, simplify the creation of reaction templates for the REACTER protocol, and increase compatibility with external visualization tools, such as VMD and OVITO. An introductory primer on the forms and use of bonded force fields is provided to motivate this new functionality and serve as an entry point for LAMMPS and OpenKIM users unfamiliar with bonded force fields. The type label framework has the potential to streamline modeling workflows that use LAMMPS by increasing the portability of software, files, and scripts for preprocessing, running, and postprocessing a molecular simulation.

Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Antiviral drug recognition and elevator-type transport motions of CNT3.

Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N4-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.

Solid-state NMR MAS CryoProbe enables structural studies of human blood protein vitronectin bound to hydroxyapatite.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.

Analogs of α-conotoxin PnIC selectively inhibit α7ß2- over α7-only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.

Spontaneous curvature generation by peptides in asymmetric bilayers.

Peptides and proteins play crucial roles in membrane remodeling by inducing spontaneous curvature. However, extracting spontaneous curvatures from simulations of asymmetric bilayers is challenging because differential stress (i.e., the difference of the leaflet surface tensions) arising from leaflet area strains can vary substantially among initial conditions. This study investigates peptide-induced spontaneous curvature δc 0 p in asymmetric bilayers consisting of a single lipid type and a peptide confined to one leaflet; δc 0 p is calculated from the Helfrich equation using the first moment of the lateral pressure tensor and an alternative expression using the differential stress. It is shown that differential stress introduced during initial system generation is effectively relaxed by equilibrating using P21 periodic boundary conditions, which allows lipids to switch leaflets across cell boundaries and equalize their chemical potentials across leaflets. This procedure leads to robust estimates of δc 0 p for the systems simulated, and is recommended when equality of chemical potentials between the leaflets is a primary consideration.

Anillin-related Mid1 as an adaptive and multimodal contractile ring anchoring protein: A simulation study.

Cytokinesis of animal and fungi cells depends crucially on the anillin scaffold proteins. Fission yeast anillin-related Mid1 anchors cytokinetic ring precursor nodes to the membrane. However, it is unclear if both of its Pleckstrin Homology (PH) and C2 C-terminal domains bind to the membrane as monomers or dimers, and if one domain plays a dominant role. We studied Mid1 membrane binding with all-atom molecular dynamics near a membrane with yeast-like lipid composition. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the disordered L3 loop of C2 in a vertical orientation, with the PH away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. Simulations of C2-PH dimers show extensive asymmetric membrane contacts. These multiple modes of binding may reflect Mid1's multiple interactions with membranes, node proteins, and ability to sustain mechanical forces.

Structural and thermodynamic framework for PIEZO1 modulation by small molecules.

Mechanosensitive PIEZO channels constitute potential pharmacological targets for multiple clinical conditions, spurring the search for potent chemical PIEZO modulators. Among them is Yoda1, a widely used synthetic small molecule PIEZO1 activator discovered through cell-based high-throughput screening. Yoda1 is thought to bind to PIEZO1's mechanosensory arm domain, sandwiched between two transmembrane regions near the channel pore. However, how the binding of Yoda1 to this region promotes channel activation remains elusive. Here, we first demonstrate that cross-linking PIEZO1 repeats A and B with disulfide bridges reduces the effects of Yoda1 in a redox-dependent manner, suggesting that Yoda1 acts by perturbing the contact between these repeats. Using molecular dynamics-based absolute binding free energy simulations, we next show that Yoda1 preferentially occupies a deeper, amphipathic binding site with higher affinity in PIEZO1 open state. Using Yoda1's binding poses in open and closed states, relative binding free energy simulations were conducted in the membrane environment, recapitulating structure-activity relationships of known Yoda1 analogs. Through virtual screening of an 8 million-compound library using computed fragment maps of the Yoda1 binding site, we subsequently identified two chemical scaffolds with agonist activity toward PIEZO1. This study supports a pharmacological model in which Yoda1 activates PIEZO1 by wedging repeats A and B, providing a structural and thermodynamic framework for the rational design of PIEZO1 modulators. Beyond PIEZO channels, the three orthogonal computational approaches employed here represent a promising path toward drug discovery in highly heterogeneous membrane protein systems.

Understanding the molecular mechanisms of odorant binding and activation of the human OR52 family.

Structural and mechanistic studies on human odorant receptors (ORs), key in olfactory signaling, are challenging because of their low surface expression in heterologous cells. The recent structure of OR51E2 bound to propionate provided molecular insight into odorant recognition, but the lack of an inactive OR structure limited understanding of the activation mechanism of ORs upon odorant binding. Here, we determined the cryo-electron microscopy structures of consensus OR52 (OR52cs), a representative of the OR52 family, in the ligand-free (apo) and octanoate-bound states. The apo structure of OR52cs reveals a large opening between transmembrane helices (TMs) 5 and 6. A comparison between the apo and active structures of OR52cs demonstrates the inward and outward movements of the extracellular and intracellular segments of TM6, respectively. These results, combined with molecular dynamics simulations and signaling assays, shed light on the molecular mechanisms of odorant binding and activation of the OR52 family.

Molecular Modeling and Simulation of the Mycobacterial Cell Envelope: From Individual Components to Cell Envelope Assemblies.

Unlike typical Gram-positive bacteria, the cell envelope of mycobacteria is unique and composed of a mycobacterial outer membrane, also known as the mycomembrane, a peptidoglycan layer, and a mycobacterial inner membrane, which is analogous to that of Gram-negative bacteria. Despite its importance, however, our understanding of this complex cell envelope is rudimentary at best. Thus, molecular modeling and simulation of such an envelope can benefit the scientific community by proposing new hypotheses about the biophysical properties of its different layers. In this Perspective, we present recent advances in molecular modeling and simulation of the mycobacterial cell envelope from individual components to cell envelope assemblies. We also show how modeling other types of cell envelopes, such as that of Escherichia coli, may help modeling part of the mycobacterial envelopes. We hope that the studies presented here are just the beginning of the road and more and more new modeling and simulation studies help us to understand crucial questions related to mycobacteria such as antibiotic resistance or bacterial survival in the host.

Accurate Force Fields for Atomistic Simulations of Oxides, Hydroxides, and Organic Hybrid Materials up to the Micrometer Scale.

The simulation of metals, oxides, and hydroxides can accelerate the design of therapeutics, alloys, catalysts, cement-based materials, ceramics, bioinspired composites, and glasses. Here we introduce the INTERFACE force field (IFF) and surface models for α-Al2O3, α-Cr2O3, α-Fe2O3, NiO, CaO, MgO, ß-Ca(OH)2, ß-Mg(OH)2, and ß-Ni(OH)2. The force field parameters are nonbonded, including atomic charges for Coulomb interactions, Lennard-Jones (LJ) potentials for van der Waals interactions with 12-6 and 9-6 options, and harmonic bond stretching for hydroxide ions. The models outperform DFT calculations and earlier atomistic models (Pedone, ReaxFF, UFF, CLAYFF) up to 2 orders of magnitude in reliability, compatibility, and interpretability due to a quantitative representation of chemical bonding consistent with other compounds across the periodic table and curated experimental data for validation. The IFF models exhibit average deviations of 0.2% in lattice parameters, <10% in surface energies (to the extent known), and 6% in bulk moduli relative to experiments. The parameters and models can be used with existing parameters for solvents, inorganic compounds, organic compounds, biomolecules, and polymers in IFF, CHARMM, CVFF, AMBER, OPLS-AA, PCFF, and COMPASS, to simulate bulk oxides, hydroxides, electrolyte interfaces, and multiphase, biological, and organic hybrid materials at length scales from atoms to micrometers. The nonbonded character of the models also enables the analysis of mixed oxides, glasses, and certain chemical reactions, and well-performing nonbonded models for silica phases, SiO2, are introduced. Automated model building is available in the CHARMM-GUI Nanomaterial Modeler. We illustrate applications of the models to predict the structure of mixed oxides, and energy barriers of ion migration, as well as binding energies of water and organic molecules in outstanding agreement with experimental data and calculations at the CCSD(T) level. Examples of model building for hydrated, pH-sensitive oxide surfaces to simulate solid-electrolyte interfaces are discussed.