Investigations of the Structure, Topology, and Interactions of the Transmembrane Domain of the Lipid-Sorting Protein p24 Being Highly Selective for Sphingomyelin-C18.

The p24 proteins play an important role in the secretory pathway where they selectively connect various cargo to other proteins, thereby being involved in the controlled assembly and disassembly of the coat protein complexes and lipid sorting. Recently, a highly selective lipid interaction motif has been identified within the p24 transmembrane domain (TMD) that recognizes the combination of the sphingomyelin headgroup and the exact length of the C18 fatty acyl chain (SM-C18). Here, we present investigations of the structure, dynamics, and sphingomyelin interactions of the p24 transmembrane region using circular dichroism, tryptophan fluorescence, and solid-state nuclear magnetic resonance (NMR) spectroscopies of the polypeptides and the surrounding lipids. Membrane insertion and/or conformation of the TMD is strongly dependent on the membrane lipid composition where the transmembrane helical insertion is strongest in the presence of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and SM-C18. By analyzing solid-state NMR angular restraints from a large number of labeled sites, we have found a tilt angle of 19° for the transmembrane helical domain at a peptide-to-lipid ratio of 1 mol %. Only minor changes in the solid-state NMR spectra are observed due to the presence of SM-C18; the only visible alterations are associated with the SM-C18 recognition motif close to the carboxy-terminal part of the hydrophobic transmembrane region in the proximity of the SM headgroup. Finally, the deuterium order parameters of POPC- d were nearly unaffected by the presence of SM-C18 or the polypeptide alone but decreased noticeably when the sphingomyelin and the polypeptide were added in combination.