A Cholesterol Dimer Stabilizes the Inactivated State of an Inward‐Rectifier Potassium Channel

Abstract

Cholesterol oligomers reside in multiple membrane protein X-ray crystal structures. Yet, there is no direct link between these oligomers and a biological function. Here we present the structural and functional details of a cholesterol dimer that stabilizes the inactivated state of an inward-rectifier potassium channel KirBac1.1. K + efflux assays confirm that high cholesterol concentration reduces K + conductance. We then determine the structure of the cholesterol-Kir-Bac1.1 complex using Xplor-NIH simulated annealing calculations driven by solid-state NMR distance measurements. These calculations identified an α-α cholesterol dimer docked to a cleft formed by adjacent subunits of the homotetrameric protein. We compare these results to coarse grain molecular dynamics simulations. This is one of the first examples of a cholesterol oligomer performing a distinct biological function and structural characterization of a conserved promiscuous lipid binding region.

2H,13C-Cholesterol for Dynamics and Structural Studies of Biological Membranes

“… 48 We recently used this procedure to produce cholesterol-rich liposomes prior to inserting KirBac 1.1 to determine the structural details of KirBac 1.1 inactivation by cholesterol. 49 Assay data within that text further established the formation of LUVs.…”

“…This sample preparation has been characterized and validated by our laboratory for different lipid mixtures, including native stain electron microscopy, which were found to be large unilamellar vesicles prior to pelleting and packing into the rotor . We recently used this procedure to produce cholesterol-rich liposomes prior to inserting KirBac 1.1 to determine the structural details of KirBac 1.1 inactivation by cholesterol . Assay data within that text further established the formation of LUVs.…”

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins

“…This can aid interpretation of structurefunction relationships. Obtain a more complete picture of lipid interactions within the context of a native-like membrane. This may reveal transient lipid interaction sites which are less likely to survive the purification strategies used in cryo-EM, as well as highlight the importance of lipid-lipid interactions, such as cholesterol stacking 26 . Quantify the kinetics of lipid binding to different sites or of multiple lipids binding to the same site. This can be used infer which sites may be more important in a biological context. Assess differences in lipid binding properties compared with related detergent densities. Check whether sterol derivates such as cholesterol-hemisuccinate, commonly used as detergents in protein purification, bind in a similar location to cholesterol in simulations.…”

“… Obtain a more complete picture of lipid interactions within the context of a native-like membrane. This may reveal transient lipid interaction sites which are less likely to survive the purification strategies used in cryo-EM, as well as highlight the importance of lipid-lipid interactions, such as cholesterol stacking 26 .  Quantify the kinetics of lipid binding to different sites or of multiple lipids binding to the same site.…”

Mutational Insight into Allosteric Regulation of Kir Channel Activity

“…MD was performed in GROMACS version 2020 using the Martini2.2 forcefield. ,, Minimization and equilibration molecular dynamics parameter (MDP) files were the default CHARMM-GUI outputs. Production made use of a custom MDP file first described by Duncan et al and more recently by Borcik et al . Minimization and equilibration were performed with the Berendsen barostat, v-rescale thermostat, and reaction-field electrostatics.…”