EXPERIMENTAL INVESTIGATION OF THE LIPID-BINDING MECHANISM OF OSH4 PROTEIN

Abstract

Recent findings show that intracellular lipid traffic between organelles primarily occurs through a non-vesicular pathway involving lipid transport proteins (LTPs) and is facilitated by areas of close apposition between two organelles so called membrane contact sites (MCS). Oxysterol-binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae serve as examples of LTPs. Osh proteins are crucial for transporting signaling lipids and are believed to form MCSs. In this study, we examined the binding mechanism of the Osh4 protein, aiming to gain a better understanding of its explicit membrane-binding mechanism.The Osh4 protein possesses an α-helical binding domain known as the amphipathic lipid-packing sensor (ALPS)-like motif. Our approach involved utilizing experimental methods to examine the biophysical interactions of both the ALPS peptide and the full-length Osh4 protein. To investigate the binding interactions of ALPS with membranes of different lipid compositions, we examined its interactions with three different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; has a zwitterionic head group) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS; has a negatively charged head group)—1:1 POPC-POPS, 4:1 POPC-POPS, and 9:1 POPC-POPS—as well as pure POPC. To understand the structural changes in ALPS and model membranes during peptide-membrane interactions, we performed a series of experimental studies. Circular dichroism (CD) was used to study the changes in the secondary structure of ALPS in different environments. The CD data indicated that the α-helical conformation of the ALPS peptide was more pronounced in the presence of POPC-POPS liposomes, especially with a higher content of POPS lipid, compared to liposomes composed entirely of POPC. This observation underscores the significant influence of anionic lipids in the facilitation of peptide folding at the membrane-water interface. X-ray diffraction was utilized to study the changes in membrane structure upon ALPS binds to it. The X-ray diffraction results showed that the ALPS peptide caused thinning of the multilayer with an increased POPS lipid ratio. This could be due to the electrostatic interaction of the positively charged Lys residue in the ALPS sequence with the anionic POPS lipid. We also studied the binding of the peptide to membranes by observing changes in the Trp fluorescence emission spectrum of ALPS upon the addition of liposomes. We observed a blue shift in the fluorescence emission maximum of Trp with higher POPS content. This suggests that the ALPS peptide was experiencing a more hydrophobic and less polar environment in the presence of the liposomes, indicating possible penetration of the peptide into the hydrocarbon region of the bilayer. The blue shifts of Trp emission in the presence of POPS liposomes were higher than those observed with POPC liposomes and suggest that the ALPS peptide binds better to charged POPS lipids, which is consistent with the X-ray diffraction data. We also conducted Trp fluorescence titration and ITC experiments to gain deeper insights into the binding affinity of the ALPS peptide to a model membrane. Using fluorescence data, we estimated the binding constant for the binding of ALPS to liposomes by performing titration measurements of vesicles with the ALPS peptide. Our analysis demonstrated that ALPS binding to 4:1 POPC-POPS lipid membranes had a Kd of 1.88 ± 0.47 μM, which corresponds to a free energy change (ΔG) of -7.82 ± 0.15 kcal/mol. Additionally, the ITC experiments performed with the same vesicles yielded a ΔG of -4.41± 0.04 kcal/mol. This result is slightly less than the ΔG value of -7.82 ± 0.15 kcal/mol obtained from fluorescence spectroscopy titration. The observed discrepancy of -3.41 kcal/mol may indicate the energy associated with the folding of the ALPS peptide. In order to understand how Osh4 forms MCSs between two membranes, we need to examine how the membranes interact with the full-length protein. The first step to achieve this is to produce the protein through recombinant protein production methods. After evaluating two different fusion tags, glutathione S-transferase (GST) and small ubiquitin-related modifier (SUMO), it was found that the SUMO tag resulted in higher protein yield and greater protein purity. Our work lays the foundation for future experiments with the full-length Osh4 protein to improve our understanding of the mechanisms of lipid transport between membranes. Our results emphasize the ALPS peptide’s selectivity for specific lipid environments, particularly its affinity for anionic lipids. We demonstrated that the presence of anionic lipids is crucial for the motif's ability to induce conformational changes upon binding to a membrane, and these conformational changes likely play a critical role in intracellular lipid trafficking and membrane organization.