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

Abstract

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...

Gating Mechanism of the Voltage-Gated Proton Channel Studied by Molecular Dynamics Simulations

“…We observed the upward displacement of the S4 helix to have a strong positive net charge in channel activation, and this is consistent with previous studies [ 14 , 15 , 16 , 17 , 18 , 20 ]. Along with the upward movement of the S4 helix, a change in the hydrogen bond network ( Figure 5 ) was observed, which is consistent with previous studies [ 14 , 16 , 17 ].…”

“…In all of the previous studies, the homology modeling of Hv1 was built based on the structures of Kv, Nav, and the Kv1.2–Kv2.1 paddle chimera as templates, with sequence identities of less than 30% [ 13 , 14 , 15 ]. Recently, voltage-dependent structural models and the inhibitor (2GBI) binding of the human Hv1 channel were reported using the homology modeling of Hv1 based on the mHv1cc crystal structure to run molecular dynamics (MD) simulations [ 16 , 17 , 18 ]. Despite the previous research attempts, the understanding of the gating mechanisms of Hv1 is still in debate, especially with the gating mechanism under the membrane potential.…”

HIFs: New arginine mimic inhibitors of the Hv1 channel with improved VSD–ligand interactions

“…We verified that the new inhibitor core structure is compatible with the 2GBI binding site ( Hong et al, 2014 ) by docking HIF within the structural model of the Hv1 VSD in the up state that was previously used for the characterization of 2GBI binding ( Geragotelis et al, 2020 ; Lim et al, 2020 ). We set the initial position of HIF so that its 2-aminoimidazole ring and the two carbon atoms at positions 4 and 5 would overlap with the corresponding moiety of bound 2GBI.…”

“…2GBI has been shown to inhibit Hv1 through an open-channel block mechanism ( Hong et al, 2013 ). Besides F150, other residues were found to interact with the ligand, including D112, S181, and R211 from the S1, S3, and S4 helices, respectively ( Chamberlin et al, 2014 ; Gianti et al, 2016 ; Hong et al, 2014 ; Lim et al, 2020 ). To develop high-affinity inhibitors based on the HIF scaffold, it is important to establish whether the same residues involved in 2GBI binding interact with HIF or whether there are different/additional molecular determinants.…”

Correction Schemes for Absolute Binding Free Energies Involving Lipid Bilayers

“… 47 Similarly, there are studies that have shown that the Rocklin correction has very little effect on the RBFE calculations. 48 This is likely best explained by the fact that the Rocklin correction mainly corrects for the long-range effects of the electrostatic interactions. The ΔΔ G DSC ( L ), which is usually the largest component in Rocklin correction, depends on the proportion of the box that is filled with water.…”