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

Abstract

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, in silico 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. In silico 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 in silico 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.

Exploring the trimerization process of a transmembrane helix with an ionizable residue by molecular dynamics simulations: a case study of transmembrane domain 5 of LMP-1

“…The ionizable residue D150 plays a key role in overcoming the entropic penalty to form oligomers, which is consistent with previous studies showing that a single polar residue (Asn, Gln, Asp, and Glu) in the transmembrane segment could mediate the formation of stable trimers by shifting its p K a s in the membrane environment. 30,56 These results also suggest an approach to the design of variants of single-span transmembrane peptide modulators with various potentials to interrupt oligomers in the bilayer.…”

“…Previous studies indicated that D150 was the key residue for TMD5 to form a trimeric structure. 25,30 Hence, the conformation of D150 was monitored, and the dihedral of the D150 side chain was measured. At the reaction coordinate of 11.15 Å, all D150s pointed to the center of the trimer (Fig.…”

“…The lumen of the TMD5 trimer frequently captures water molecules, which causes a varied number of hydrogen bonds 28 and an asymmetric distance between every two monomers. 30 This could be the other factor contributing to the asymmetric environment for D150. With increasing reaction coordinates, the dihedrals of the D150 side chains showed a broader distribution (Fig.…”

“…The initial structure of the protonated TMD5 trimer was equilibrated in 200 ns simulations with several repeats in our previous study. 30 Briefly, the TMD5 trimer was placed in a 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) bilayer and solvated by the TIP3P water model with 150 mM KCl in a box with a size of 100 Å × 100 Å × 88 Å (297 lipids and 14868 water molecules) using the CHARMM–GUI membrane builder. 31 The CHARMM C36m and CHARMM C36 force fields were applied to proteins and lipids, respectively.…”

“…26–28 Further investigation showed that the D150-protonated TMD5 trimer was stable, whereas unprotonated D150 created bending in the helices that distorted the trimeric structure. 30 Herein, TMD5 of LPM-1 from EBV was used as a prototype model to investigate the trimerization process of the transmembrane segment with ionizable residues. The behavior of the transmembrane helix and the p K a shift of ionizable residues during the trimerization process were inspected via conventional molecular dynamics simulations and constant-pH molecular dynamics (CpHMD) simulations.…”