(700f) PIP Binding and Membrane Attachment of a Protein Involved in Intracellular Transport of Sterols

Osh4 is an oxysterol binding protein (OSBP) homologue found in yeast that is essential for the intracellular transport of sterols and for cell life. It has been proposed that Osh4 acts as a lipid transport protein, capable of binding a single sterol within a hydrophobic binding pocket and carrying it from the endoplasmic reticulum to the plasma membrane (PM). Phosphoinositides (PIs) are thought to stimulate sterol transfer by binding to a certain region of the Osh4 protein surface. While larger, human homologues interact with PIPs via a Pleckstrin homology (PH) domain, smaller OSBP-related proteins (ORPs) do not contain a PH domain, and may interact with PIPs through multiple mechanisms.

In order to study how the Osh4 protein attaches to the PM, possible lipid binding sites were investigated through the use of blind docking techniques. Model ligand compounds for phosphocholine (PC) and two PI [PI(4,5)P2 and PI(3,4,5)P3] head groups were docked against several conformational snapshots of the Osh4 proteins surface to determine possible regions favorable to interactions with PM lipids. These conformational snapshots were taken from two 25-ns molecular dynamics (MD) simulations of the Osh4 protein complexed with ergosterol and two complexed with 25-hydroxycholesterol. The PIP models frequently docked to a lysine-rich region on the side of the protein near the protein's flexible lid subdomain. This region is also bounded by a flexible surface loop that is believed to be important for Osh4-membrane binding.

Osh4-membrane interaction was also investigated through MD simulations of a combined membrane and protein system. A model yeast PM containing five different lipid types and ergosterol was constructed using CHARMM-GUI and equilibrated using 60 ns of MD simulation. Key residues of the Osh4 protein that were identified during the blind docking tests were placed in close proximity to the membrane surface. Ultimately, understanding how Osh4 attaches to the PM will lead to a clear understanding on how this protein transports sterols in vivo.