Electronic Polarizability Tunes the Function of the Human Bestrophin 1 Cl- Channel

Abstract

Mechanisms of anion permeation within ion channels and nanopores remain poorly understood. Recent cryo-electron microscopy structures of the human bestrophin 1 Cl- channel (hBest1) provide an opportunity to evaluate ion interactions predicted by molecular dynamics (MD) simulations against experimental observations. Here, we implement the fully polarizable forcefield AMOEBA in MD simulations on different conformations of hBest1. This forcefield models multipole moments up to the quadrupole; therefore, it captures induced dipole and anion-π interactions. We show that key biophysical properties of the channel can only be simulated when electronic polarization is included in the molecular models and that Cl- permeation through the neck of the pore is achieved through hydrophobic solvation concomitant with partial ion dehydration. Furthermore, we demonstrate how such polarizable simulations can help determine the identity of ion-like densities within high-resolution cryo-EM structures and that neglecting polarization places Cl- at positions that do not correspond with their experimentally resolved location. Overall, our results demonstrate the importance of including electronic polarization in realistic and physically accurate models of biological systems, especially channels and pores that selectively permeate anions.

Keywords: Bestrophin channels; Induced polarization; anion permeation; anion-π; chloride selectivity; molecular dynamics.

Figure 1:. Cryo-EM structures of hBest1 in the open and “partially open” states.

A Open state hBest1 (PDB ID 8D1O) and D partially open state hBest1 (PDB ID 8D1K) structures visualized with ion permeation pathway in white. The transmembrane neck region of the B open state and E partially open state structures, with ion permeation pathway colored by hydrophobicity with pale brown corresponding to maximum hydrophobicity and green corresponding to maximum hydrophilicity. Top-down view of the C open state neck and F partially open state neck. G Pore radius profiles of the open, partially open and closed (PDB ID 8D1I) states.

Figure 2:. Cl⁻ binding site analysis.

A Close-up representation of neck residues (I76, F80 and F84) from a single chain highlighted in licorice representation of the open state structure (PDB ID 8D1O). The cryo-EM densities are shown as a mesh representation in purple. A panel is zoomed in on the non-protein ion-like density in proximity to F80 labelled as X739, which corresponds to the water molecule HOH739 in the PDB. B The trajectories of water (cyan), Na+ (green) and Cl⁻ (orange) ions in z-coordinates as a function of time within the neck region of the pore. The plot indicates that Cl⁻ tends to cluster in a region between I76 and F80. The green dashed line represents the location of X739 from the experimental structure. AMOEBA forcefield simulations: Top-down view of the protein fragment in C AMOEBA (orange) or F c36m (magenta) aligned to the full protein structure (cyan). The location of X739 is indicated by the green sphere. The top detected binding pose identified with PyLipID shows D significant overlap between the AMOEBA Cl⁻ (orange sphere) and X739, indicating this site likely functions as a Cl⁻ binding site or G the c36m Cl⁻ (magenta sphere) shares no overlap with X739. E, H A rotated view showing the distance of the Cl⁻ and X739 to the backbone N of F80 in the experimental structure.

Figure 3:. The role of water in Clˉ permeation in the open state AMOEBA simulations.

A Cross-section schematic of the protein fragment of the open state bestrophin (PDB ID 8D1O) with the neck residues illustrated in licorice representation. B The pore radius profile of the open state fragment. The outer grey dashed lines represent the extent of the protein and the central lines indicate the z-location of the neck residues where z is the distance down the axis of the pore and z = 0 corresponds to the C_α of F80. C The first hydration shell of Clˉ as a function of z. The shaded orange region represents the standard error in hydration number at a given z. Snapshots exemplifying the first hydration shell (blue spheres) of Cl⁻ (orange spheres) in D the bulk-like water regime within the protein fragment. E The partial loss of hydration shell due to anion-π interactions with the aromatic rings of F80 rotated inwards towards the pore axis. F The partial loss of Clˉ hydration due to anion-π interactions with the aromatic rings of F80 oriented outwards as in the experimental structure conformation.

Figure 4:. Analysis of the partially open state

Trajectories of water (cyan), sodium (green) and Cl⁻ (orange) ions in z-coordinates as a function of time within the neck region of the partially open state pore (PDB ID 8D1K) using A the AMOEBA forcefield or B c36m forcefield. The red box in A highlights a Cl⁻ that can occupy locations above F84 in the neck whereas nearly no ions can be seen in B with c36m. C Bottom-up view of F84 of the partially open state neck. The volume that Cl⁻ may occupy over the simulation is represented by the orange mesh. D Side-view of the neck region. F84 of the neck may accommodate for Cl⁻; however, the neck is not permeable to Cl⁻. E Bottom-up view of F84. Cl⁻ can be seen to interact with the aromatic ring through edgewise anion-π interactions. The coordinating atoms within the first solvation shell of the Cl⁻ (orange sphere) are represented as spheres.