Voltage-dependent structural models of the human Hv1 proton channel from long-timescale molecular dynamics simulations

Geragotelis

et al. 2020

Sci Rep

Self Cite

Hv1 is a voltage-gated proton channel whose main function is to facilitate extrusion of protons from the cell. The development of effective channel blockers for Hv1 can lead to new therapeutics for the treatment of maladies related to Hv1 dysfunction. Although the mechanism of proton permeation in Hv1 remains to be elucidated, a series of small molecules have been discovered to inhibit Hv1. Here, we computed relative binding free energies of a prototypical Hv1 blocker on a model of human Hv1 in an open state. We used alchemical free energy perturbation techniques based on atomistic molecular dynamics simulations. The results support our proposed open state model and shed light on the preferred tautomeric state of the channel blocker. This work lays the groundwork for future studies on adapting the blocker molecule for more effective inhibition of Hv1. The flux of ions across the cell membrane is regulated by a variety of ion channels. Hv1 is a voltage-dependent ion channel whose main function is to conduct protons through the cell membrane. Like voltage-dependent metal ion channels, the activation of Hv1 is regulated by voltage-sensing domains (VSDs), which are transmembrane modular units that detect changes in the membrane potential. However, in contrast to voltage-dependent metal ion channels, Hv1 lacks a separate pore domain as the VSD is sufficient for proton permeation in addition to mediating channel gating 1-3. The action of Hv1 contributes to physiological processes such as the production of reactive oxygen species, the bioluminescence of dinoflagellates, and the maturation of human sperm 4-6. Hv1 expression was found to be selectively enhanced in metastatic relative to non-metastatic breast tissue and inhibition of Hv1 was shown to reduce cancer metastasis and tumor development 7-9. More generally, the development of inhibitors to block the Hv1 channel may also lead to therapeutic benefit for other Hv1-related maladies, including allergies 10,11 or exacerbated brain damage in ischemic stroke 12. The full structure of human Hv1 has not yet been experimentally determined. Human Hv1 is a homodimer, but each monomer has its own pore and can function independently 13,14. We recently developed an atomistic model of the human Hv1 VSD open state using molecular dynamics (MD) simulations on the microsecond timescale in a hydrated lipid bilayer under an applied membrane potential 15. As discussed in Geragotelis et al. 15 , there exist other proposed structures for the human Hv1 open state 16-20 , which differ in the way they were generated or the templates they were based upon. These other structures were either modeled from crystallographic structures of voltage-gated ion channels which do not contain a native permeation pathway through the VSD 16-18,20 , or they have alternative VSD conformations from restraints applied during MD simulations 19. Our structure was modeled starting from the crystal structure of a chimeric construct based on mouse Hv1 in a putative closed state 21 from which we generated closed and...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Positioning Of 2gbi Tautomers Within the Hv1 Open Statementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Insights on small molecule binding to the Hv1 proton channel from free energy calculations with molecular dynamics simulations

Geragotelis

et al. 2020

Sci Rep

Self Cite

“…There is precedence for the facilitation of ion permeation across the lipid bilayer by similar domains from potassium channels, as the isolated voltage-sensing domain (VSD) of the Shaker Kv channel has been shown to form a cation selective pore 32 . Voltage-dependent permeation of protons was also shown for the ion channel HV1 33,34 , which has only four transmembrane helices in a VSD-like arrangement (S1-S4) with a predicted hydrated pathway 33 . The individual S1-S4 subunits of TRPY1 coupled to the TRP-CTD linker and the CH1 helix featuring several charged residues at its N-terminus (Lys514 to Asp518), may function in a similar manner, explaining the observed water permeation.…”

Section: Resultsmentioning

confidence: 81%

Structure of the ancestral TRPY1 channel fromSaccharomyces cerevisiaereveals mechanisms of modulation by lipids and calcium

Ahmed

Nisler

Fluck

et al. 2020

Preprint

Transient Receptor Potential (TRP) channels have evolved in eukaryotes to control various cellular functions in response to a wide variety of chemical and physical stimuli. This large and diverse family of channels emerged in fungi as mechanosensitive osmoregulators. The Saccharomyces cerevisiae vacuolar TRP yeast 1 (TRPY1) is the most studied TRP channel from fungi, but the molecular details of channel modulation remain elusive so far. Here, we describe the full-length cryo-electron microscopy structure of TRPY1 at 3.1 A resolution. The structure reveals a distinctive architecture for TRPY1 among all eukaryotic TRP channels with an evolutionarily conserved and archetypical transmembrane domain, but distinct structural folds for the cytosolic N- and C-termini. We identified the inhibitory phosphatidylinositol 3‐phosphate (PI(3)P) lipid binding site, which sheds light into the lipid modulation of TRPY1 in the vacuolar membrane. The structure also exhibited two Ca2+-binding sites: one in the cytosolic side, implicated in channel activation, and the other in the vacuolar lumen side, involved in channel inhibition. These findings, together with data from molecular dynamics simulations, provide structural insights into the basis of TRPY1 channel modulation by lipids and calcium.

Trapped Pore Waters in the Open Proton Channel H_V1

Boytsov

Brescia

Chaves

et al. 2023

Small

to H V 1 gating. [3] Its structure is homologous to classical voltage-gated channels' voltage-sensing domain (VSD). [4] Experimentally, exclusively closed structures are available -a crystal structure of a chimera between mouse H V 1 and Ciona intestinalis voltage-sensing phosphatase [5] and an NMR structure of the human H V 1. [6] Also, electron paramagnetic resonance (EPR) spectroscopy data decipher the resting structure of H V 1. [7] An AlphaFold structure has been recently added (Figure 1). AlphaFold uses a machine-learning approach. [8] Its deep learning algorithm exploits physical and biological knowledge about protein structure -mainly gained by X-ray crystallography, cryo-electron microscopy, or NMR. None of these technical approaches allows the application of membrane voltage, a prerequisite to attain the open channel configuration of voltage-sensitive channels. Since Hv1 belongs to this class of channels, the AlphaFold structure reflects, in all likelihood, the closed channel structure.The analysis of the AlphaFold structure corroborates the conclusion (Figure 1A,B). It shows three positively charged arginine residues in the membrane-spanning part of the 4th helix (S4) responsible for Hv1's voltage sensitivity. They may interact with three negatively charged aspartate residues on the first (S1) and third helices (S3). The AlphaFold structure suggests the following Coulombic interactions: R205 -D112 (on S1), R211 -D185 (on S3) and R208 -D174 (on S3). There is consensus that i) D174 is part of the intracellular electrostatic network [9,10] and ii) D174's salt bridge with R208 stabilizes the closed, resting-state conformation. [11] The open channel's structure has solely been computed as homology models. [12][13][14][15][16][17][18] One of the most recent models reflects the experimentally estimated gating charge suggesting that the transition from the closed to the open conformation may be correctly captured. [17] Yet, the closed conformations of the homology model (Figure S1, Supporting Information) and AlphaFold (Figure 1A,B) differ in pore diameter (Figure 1C). While the former is large enough to allow water molecules to pass, the latter is much too narrow. The observation suggests that an assessment of H V 1's unitary water permeability, p f , may be used as a diagnostic tool. By undertaking water flux measurements through purified and reconstituted H V 1 channels in The voltage-gated proton channel, H V 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10 −12 cm 3 s −1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does no...