Proposed dual membrane contact with full-length Osh4

Abstract

Membrane contacts sites (MCSs) play important roles in lipid trafficking across cellular compartments and maintain the widespread structural diversity of organelles. We have utilized microsecond long all-atom (AA) molecular dynamics (MD) simulations and enhanced sampling techniques to unravel the MCS structure targeting by yeast oxysterol binding protein (Osh4) in an environment that mimics the interface of membranes with an increased proportion of anionic lipids using CHARMM36m forcefield with additional CUFIX parameters for lipid-protein electrostatic interactions. In a dual-membrane environment, unbiased MD simulations show that Osh4 briefly interacts with both membranes, before aligning itself with a single membrane, adopting a β-crease-bound conformation similar to observations in a single-membrane scenario. Targeted molecular dynamics simulations followed by microsecond-long AA MD simulations have revealed a distinctive dual-membrane bound state of Osh4 at MCS, wherein the protein interacts with the lower membrane via the β-crease surface, featuring its PHE-239 residue positioned below the phosphate plane of membrane, while concurrently establishing contact with the opposite membrane through the extended α6-α7 region. Osh4 maintains these dual membrane contacts simultaneously over the course of microsecond-long MD simulations. Moreover, binding energy calculations highlighted the essential roles played by the phenylalanine loop and the α6 helix in dynamically stabilizing dual-membrane bound state of Osh4 at MCS. Our computational findings were corroborated through frequency of contact analysis, showcasing excellent agreement with past experimental cross-linking data. Our computational study reveals a dual-membrane bound conformation of Osh4, providing insights into protein-membrane interactions at membrane contact sites and their relevance to lipid transfer processes.