A novel model of the dimeric human HV1 channel reveals a putative ATP binding site.

LLANOS, MANUEL; MARTÍN, PEDRO; ENRIQUE, NICOLÁS JORGE; FELICE, JUAN IGNACIO; ASUAJE, AGUSTÍN; GAVERNET, LUCIANA; MILESI, VERÓNICA; VENTURA, CLARA

Encuentro; 65th Biophysical Society Annual Meeting; 2021

The Biophysical Society

A multi-template model of the voltage-gated proton channel(hHv1) was developed by combining the recently solved NMR structural ensembleof the hHv1 (PDBID: 5OQK) with the coiled-coil C-terminal domain (PDBID: 3A2A),bridging them through the chimeric mHv1cc structure (PDBID: 3WKV). The modelwas built and refined as a dimer within Rosetta?s Membrane framework, using thesymmetry extracted from the coiled-coil and including experimentally derivedconstraints during the relaxation.The dimeric model was then subjected to extensive GaussianAccelerated Molecular Dynamics simulations in a POPC bilayer, andrepresentative snapshots were extracted from the trajectory by clusteringanalysis. Blind docking simulations of an ATP molecule on this structuralensemble revealed a putative ATP binding site located on the intracellularportion of the permeation pathway, below R3 (ARG211). The same region wasidentified by two sequence-based ATP binding site predictors: ATPSite andATPint; and a third meta-predictor which combines sequence and structure-basedfeatures: ATPBind.According tothe in-silico predictions, we found that whole-cell hHv1 current measured inpresence of intracellular 3 mM ATP (patch-clamp, Jurkat T cells, n=7) showed ahigher amplitude (148.3±27.0 vs 35.6±4.6 pA at 20 mV p<0.05) and ahyperpolarized Vthreshold  (-25.7±2.0 vs-12.0±3.7 mV, p<0.05), compared to control cells (without ATP, n=5).The combination of several in-silico methodologies allowedus to develop a novel model of dimeric hHv1 channel, and predict its directmodulation by ATP. Moreover, experimental results confirmed that ATP is able toactivate the channel. In a physiological context, this ATP-mediated modulationcould integrate the cell metabolic state with the H+ efflux,especially in cells where hHv1 channels are relevant for pH regulation (i.e.pancreatic 𝜷-cells, cancer cells, immune cells).