MD simulations for publication "­­­TRPV4-Rho GTPase complex structures reveal ­­mechanisms of gating and disease" on Nature Communications. Initial and last system snapshots of 8 simulation systems are provided: 1) GSK279-TRPV4 in GSK279 pose I, 2) GSK279-TRPV4 in GSK279 pose II, 3) GSK279-TRPV4-RhoA-GDP in GSK279 pose I, 4) GSK101-TRPV4, 5) GSK101-TRPV4-RhoA-GTP, 6) 4α-PDD-TRPV4 in 4α-PDD pose I, 7) 4α-PDD-TRPV4 in 4α-PDD pose II, 8) 4α-PDD-TRPV4 in 4α-PDD pose III.

Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.

. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the apo, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that the interaction strength between TRPV4 and RhoA tunes TRPV4-mediated calcium homeostasis and actin remodeling, and that disruption of TRPV4-RhoA interactions leads to TRPV4-related neuromuscular disease, findings that will guide TRPV4 therapeutics development.

Single particle cryo-EM often yields multiple protein conformations within a single dataset, but experimentally deducing the temporal relationship of these conformers within a conformational trajectory is not trivial. Here, we use thermal titration methods and cryo-EM in an attempt to obtain temporal resolution of the conformational trajectory of the vanilloid receptor TRPV1 with resiniferatoxin (RTx) bound. Based on our cryo-EM ensemble analysis, RTx binding to TRPV1 appears to induce intracellular gate opening first, followed by selectivity filter dilation, then pore loop rearrangement to reach the final open state. This apparent conformational wave likely arises from the concerted, stepwise, additive structural changes of TRPV1 over many subdomains. Greater understanding of the RTx-mediated long-range allostery of TRPV1 could help further the therapeutic potential of RTx, which is a promising drug candidate for pain relief associated with advanced cancer or knee arthritis.

MD simulations for publication "­­­TRPV4-Rho GTPase complex structures reveal ­­mechanisms of gating and disease" on Nature Communications. Initial and last system snapshots of 8 simulation systems are provided: 1) GSK279-TRPV4 in GSK279 pose I, 2) GSK279-TRPV4 in GSK279 pose II, 3) GSK279-TRPV4-RhoA-GDP in GSK279 pose I, 4) GSK101-TRPV4, 5) GSK101-TRPV4-RhoA-GTP, 6) 4α-PDD-TRPV4 in 4α-PDD pose I, 7) 4α-PDD-TRPV4 in 4α-PDD pose II, 8) 4α-PDD-TRPV4 in 4α-PDD pose III.

Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.

. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the apo, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that the interaction strength between TRPV4 and RhoA tunes TRPV4-mediated calcium homeostasis and actin remodeling, and that disruption of TRPV4-RhoA interactions leads to TRPV4-related neuromuscular disease, findings that will guide TRPV4 therapeutics development.

Single particle cryo-EM often yields multiple protein conformations within a single dataset, but experimentally deducing the temporal relationship of these conformers within a conformational trajectory is not trivial. Here, we use thermal titration methods and cryo-EM in an attempt to obtain temporal resolution of the conformational trajectory of the vanilloid receptor TRPV1 with resiniferatoxin (RTx) bound. Based on our cryo-EM ensemble analysis, RTx binding to TRPV1 appears to induce intracellular gate opening first, followed by selectivity filter dilation, then pore loop rearrangement to reach the final open state. This apparent conformational wave likely arises from the concerted, stepwise, additive structural changes of TRPV1 over many subdomains. Greater understanding of the RTx-mediated long-range allostery of TRPV1 could help further the therapeutic potential of RTx, which is a promising drug candidate for pain relief associated with advanced cancer or knee arthritis.

Abstract The ClpP serine peptidase is a tetradecameric degradation machine involved in many physiological processes. It becomes a competent ATP-dependent protease with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause dysregulation and activation of ClpP without ATPases, and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed the structural details, conformational changes, and activation mechanism. Although product release by the side exit pores has been proposed, the detailed driving force for product release remains elusive. Here, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures revealed various conformational states at different pH conditions. To understand the conformational change for product release, we investigated the relationship between substrate hydrolysis and the pH lowering process. Our data, together with previous findings, provide insight into the molecular mechanism of product release by ClpP self-compartmentalizing protease.