Dissecting Role of Charged Residue from Transmembrane Domain 5 of Latent Membrane Protein 1 via Simulations and Wet-Lab Experiments.

Charged residues are frequently found in the transmembrane segments of membrane proteins, which reside in the hydrophobic bilayer environment. Charged residues are critical for the function of membrane protein. However, studies of their role in protein oligomerization are limited. By taking the fifth transmembrane domain (TMD5) of latent membrane protein 1 from the Epstein-Barr virus as a prototype model, simulations and wet-lab experiments were performed to investigate how the charged states affect transmembrane domain oligomerization. Molecular dynamics (MD) simulations showed that the D150-protonated TMD5 trimer was stable, whereas unprotonated D150 created bends in the helices which distort the trimeric structure. D150 was mutated to asparagine to mimic the protonated D150 in TMD5, and the MD simulations of different D150N TMD5 trimers supported that the protonation state of D150 was critical for the trimerization of TMD5. mutations found that D150N TMD5 preferred to interact with TMD5 to form the heterotrimer (1 D150N TMD5:2 protonated TMD5s) rather than the heterotrimer (2 D150N TMD5s:1 protonated TMD5). D150R TMD5 interacted with TMD5 to form the heterotrimer (1 D150R TMD5:2 protonated TMD5). These results imply that D150N TMD5 and D150R TMD5 peptides may be probes for disrupting TMD5 trimerization, which was supported by the dominant-negative ToxR assay in bacterial membranes. In all, this study elucidates the role of charged residues at the membrane milieu in membrane protein oligomerization and provides insight into the development of oligomerization-regulating peptides for modulating transmembrane domain lateral interactions.