development, combining different levels of theory to increase accuracy, aiming to connect chemical and molecular changes to macroscopic observables. In this review, we outline biomolecular simulation methods and highlight examples of its application to investigate questions in biology. enzyme, membrane, molecular dynamics, multiscale, protein, QM/MM 1 | INTRODUCTION Biomolecular simulations are now making significant contributions to a wide variety of problems in drug discovery, drug development, biocatalysis, biotechnology, nanotechnology, chemical biology, and medicine. Biomolecular simulation is a rapidly growing field in scale and impact, increasingly demonstrating its worth in understanding mechanisms and analyzing activities, and contributing to the design of drugs and biocatalysts. Physics-based simulations complement experiments in building a molecular-level understanding of biology: They can test hypotheses and interpret and analyze experimental data in terms of interactions at the atomic level. Different types of simulation techniques have been developed, which are applicable to a range of different problems in biomolecular science. Simulations have already shown their worth in helping to analyze how enzymes catalyze biochemical reactions, and how proteins adopt their functional structures, for example, within cell membranes. They contribute to the design of drugs and catalysts, and in understanding the molecular basis of disease. Simulations have played a key role in developing the conceptual framework now at the heart of biomolecular science: that the dynamics of biological molecules is central to their function. Developing methods from chemical physics and computational science will open exciting new opportunities in biomolecular science, including in drug design and development, biotechnology, and biocatalysis. With high-performance computing resources, large-scale atomistic simulations of biological machines such as the ribosome, proton pumps and motors, membrane receptor complexes, and even whole viruses have become possible. Useful simulations of smaller systems can be carried out with desktop resources, thanks to developments allowing, for example, graphics processing units (GPUs) to be used. A particular challenge across the field is the integration of simulations crossing the span of length-and timescales as different types of simulation method are required for different types of problems. 1 Biomolecular systems pose fundamental scientific challenges (e.g., protein folding, enzyme catalysis, gene regulation, disease mechanisms, and antimicrobial resistance) and are at the heart of many advanced technological developments (drug discovery, biotechnology, biocatalysis, biomaterials, and genetic engineering). Biomolecular systems are inherently complex and pose significant challenges in modeling. An essential underlying paradigm is the need to consider biomolecular ensembles and their dynamics, rather than simply static biomolecular structures to understand and predict their behavior and propertie...

Highlights d MD is used to refine problematic regions of the open state of the glycine receptor d We functionally annotate it as open and selective for chloride ions d The open state is stabilized by the 9 0 residues entering conserved hydrophobic pockets d The protocol can be more broadly applied to all members of the Cys-loop family

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The neuropeptide Substance P (SP) is important in pain and inflammation. SP activates the neurokinin-1 receptor (NK1R) to signal via G q and G s proteins. Neurokinin A also activates NK1R, but leads to selective G q signaling. How two stimuli yield distinct G protein signaling at the same G protein-coupled receptor remains unclear. We determined cryo-EM structures of active NK1R bound to SP or the G q -biased peptide SP6–11. Peptide interactions deep within NK1R are critical for receptor activation. Conversely, interactions between SP and NK1R extracellular loops are required for potent G s signaling but not G q signaling. Molecular dynamics simulations showed that these superficial contacts restrict SP flexibility. SP6–11, which lacks these interactions, is dynamic while bound to NK1R. Structural dynamics of NK1R agonists therefore depend on interactions with the receptor extracellular loops and regulate G protein signaling selectivity. Similar interactions between other neuropeptides and their cognate receptors may tune intracellular signaling.

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor’s conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.

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Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules.

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that the lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor’s conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.Author SummaryIon channels are proteins that control the flow of ions into the cell. The family of ion channels known as the pentameric ligand gated ion channels (pLGICS) open in response to the binding of a neurotransmitter, moving the channel from a resting state to an open state. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. It is also known that the response of pLGICs can also be modified by different types of lipid found within the membrane itself but exactly how is unclear. Here, we used a realistic model of a neuronal membrane and performed molecular dynamics simulations to show various lipid-protein interactions that are dependent on the channel state. Our work also reveals previously unconsidered protein-lipid interactions at a key junction of the channel known to be critical for the transmission of the opening process. We also demonstrate that cholesterol interacts with the protein at a site already known to bind to another compound that modulates the channel, called ivermectin. The work should be useful for future drug design.