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

Christopher Aisenbrey,Burkhard Bechinger,Patricia Kemayo-Koumkoua,Evgeniy S Salnikov,Elise Glattard

doi:10.1021/acs.biochem.9b00375

Abstract

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- d31 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.

Highlights

Investigations of the Structure, Topology, and Interactions of the Transmembrane Domain of the Lipid-Sorting Protein p24 Being
To perform structural investigations of the p24 transmembrane domain, a peptide encompassing the hydrophobic membrane anchor and the SM-C18 recognition motif was prepared by solid phase peptide synthesis
In the work presented here, the transmembrane domain of p24 was investigated by circular dichroism (CD), fluorescence, and solid-state nuclear magnetic resonance (NMR)

Summary

Introduction

Investigations of the Structure, Topology, and Interactions of the Transmembrane Domain of the Lipid-Sorting Protein p24 Being. 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. 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). The FRET signal was considerably weaker in the presence of sphingomyelins with different chain lengths or for the corresponding phosphatidylcholine (PC-C18:5) indicating a highly specific he secretory pathway is essential in the synthesis, transport, and processing of proteins and bioactive molecules and plays a key role in intercellular communication.[1] Thereby, it is vitally important and can be linked to a variety of diseases. During the early secretory pathway, the combination of anteriograde and retrograde transport by vesicles covered by coat protein I and II (COPI- and COPIIcoated transport vesicles, respectively) and sorting ensure a controlled and tunable system assuring the correct amount, processing, and destination of proteins and lipids in each compartment.[1,2]

Methods

Results

Discussion

Conclusion