Mechanism for membrane contact formation with full-length Osh4 in dual-membrane environment.

Abstract

Membrane contacts sites (MCSs) play important roles in lipid trafficking across cellular compartments and maintain the widespread structural diversity of organelles. Osh4 is the most abundantly expressed oxysterol binding protein (Osh) in yeast and facilitates the sterol and lipid transport via a non-vesicular pathway between cellular organelles. The functional mechanism of Osh4 in lipid transport is elusive as several experimental studies have suggested that Osh proteins operate at MCSs and shuttle lipids between membranes in a direct fashion or via diffusing through cytoplasm. We have utilized microsecond long all-atom molecular dynamics simulations and enhanced sampling techniques to unravel the functional mechanism of Osh4 protein in dual-membrane environment mimicking the interface of ER and TGN membranes using CHARMM36m forcefield with additional CUFIX parameters for lipid-protein electrostatic interactions. In dual-membrane environment, unbiased MD simulations indicate that Osh4 initially rotates approaching a single membrane and eventually establishes contact with that membrane yielding a β-crease membrane bound conformation similar to our previous observation based on single-membrane approach. We have further used targeted MD simulations based on our past work with α6-α7 region of Osh4 to accelerate the conformational transition of Osh4 enabling simultaneous binding on two opposing membranes at MCS. Interestingly, we have identified a bound state of Osh4 at MCS where the protein is interacting with the lower membrane through the β-crease surface with its PHE-239 residue located below the membrane phosphate plane and other part of the protein is simultaneously establishing contact with the opposite membrane though extended α6-α7 region. These dual membrane contacts are maintained concurrently throughout microsecond long MD simulations. Nevertheless, Osh4 structural determination at MCS utilizing NMR and cryoEM techniques are planned to support our computational findings and shed light on the functional mechanism of Osh4 at MCS.