(505c) Investigating the Membrane-Binding Mechanism of the ALPS Peptide of Osh4

Recent findings show that intracellular lipid traffic between organelles primarily occurs through a non-vesicular pathway involving lipid transport proteins (LTPs), facilitated by membrane contact sites (MCS). Oxysterol binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae serve as an example of LTPs that play an essential role in transporting signaling lipids, and we investigated the binding mechanism of the Osh4 protein.

The Osh4 protein possesses a 25 amino-acid α-helical motif known as the amphipathic lipid-packing sensor (ALPS)-like motif. To study the binding interactions of the ALPS peptide with membranes of different lipid compositions, we looked at its interactions with four different 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC ; zwitterionic choline group) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS; negatively charged phosphoserine group) mixtures: 50% POPS-50% POPC, 20% POPS-80% POPC, 10% POPS- 90% POPC, and 0% POPS-100% POPC. The X-ray diffraction (XRD) results at 93% relative humidity and 25 °C indicated that the incorporation of the ALPS peptide caused more thinning of multilayer with higher POPS lipid ratio. This observation could be due to the electrostatic interaction of the positively charged Lys15 residue in the ALPS peptide with the anionic POPS lipid.

We also investigated the binding of the peptide to membranes by recording changes in the Trp fluorescence emission spectrum of ALPS upon the addition of liposomes. The blue shift of the fluorescence emission maximum of Trp increased at higher POPS:POPC ratios. The blue shift suggests that the ALPS peptide was experiencing a more hydrophobic and less polar environment in the presence of the liposomes, which could indicate the penetration of the peptide into the hydrocarbon region of the bilayer. The higher blue shifts in the presence of POPS liposomes than POPC liposomes is consistent with the XRD data and suggests that the ALPS peptide binds better to charged POPS lipids.

To gain deeper insights into the binding dynamics of the ALPS peptide, we conducted all-atom molecular dynamics simulations. The simulations demonstrated a slightly elevated level of hydrogen bonding in a 20% POPS-80% POPC membrane compared to a homogeneous 100% POPC membrane. This observation aligns with the fluorescence and XRD experiments, indicating that the ALPS peptide exhibits a preference for binding in the presence of charged POPS lipids.

With the improved understanding of the interaction of the ALPS motif peptide with lipid membranes, we are now studying the interaction of the full-length Osh4 protein to develop a more complete understanding of its ability to form MCSs.