Specificity in Transmembrane Helix-Helix Interactions Mediated by Aromatic Residues

Abstract

Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through -interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458 -15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM selfassembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity.Receptor self-assembly is a central process in a variety of signal transduction cascades. This assembly is mainly mediated by the extracellular or the intracellular domains. However, considerable data have been accumulated concerning the causal involvement of the transmembrane (TM) 2 domains in this process as well (1-5). In contrast to the soluble regions of membrane proteins, our knowledge of the factors that control protein-protein interactions and recognition of the membraneembedded domains is still limited.To date, the non-covalent association of native TM domains was reported to be mediated by (i) a heptad motif of leucines through their side chain residues packing interaction (6); (ii) a GXXXG motif, which was first found in the glycophorin A TM domain (4, 8, 9); or (iii) polar residues through the formation of hydrogen bonds (10 -14). However, the involvement of additional motifs or key factors that may mediate protein-protein interactions within the membrane merit further investigation.Examination of the assembly of soluble proteins reveals that aromatic residues serve as key structural elements that mediate the molecular recognition and the self-assembly of amyloid polypeptides as well as bacterial toxins and several proteins such as acetylcholinesterase (15)(16)(17)(18)(19)(20). The interactions are formed between the planar aromatic rings and are referred to as -interactions (21-23). Even a single mutation of aromatic amino acid in the sequence of the short amyloid peptide abolishes the ability of the peptide to form amyloid fibri...

A Unique Phenylalanine in the Transmembrane Domain Strengthens Homodimerization of the Syndecan-2 Transmembrane Domain and Functionally Regulates Syndecan-2

“…Moreover, the presence of a phenylalanine in addition to the GXXXG motif enhances the self-association of the GpA TMD (8). Several other reports have confirmed that aromatic residues, either alone or near the GXXXG motif, can stabilize the TMD helix dimerization of membrane proteins (8,29,30 ). Therefore, we speculated that, as in the other studied receptors, phenylalanine might be involved in regulating the interaction strength of the syndecan TMDs.…”

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

“…To further pursue this hypothesis, we applied site-directed mutagenesis to the DiHcRED-MAL chimera. Mutagenesis and deletions were carried out according to the following strategies: 1) deletion of a single amino acid in each TMD or combinations of single amino acid deletions in two, three, or all four TMDs; and 2) mutagenesis of bulky amino acids within the XX intramembrane protein-protein interaction motif (Sal-Man et al, 2007). The locations of the key mutations and deletions are shown in the scheme in Figure 5a.…”

“…This lends support to the premise that OC formation, as well as lateral concentration of lipids, are mediated by interactions within the hydrophobic core of the membrane between MAL TMDs and the lipid fatty acyl tails. Moreover, the exclusive effect of shortening two TMDs on lipid concentration but not on OC formation suggests that OC formation is at least in part associated with MAL's intrinsic propensity for self-oligomerization, mediated by the recently characterized XX motif (Sal-Man et al, 2007). Consequently, we sought to substantiate MAL's propensity for self-oligomerization in epithelial cell membranes as well as independently of the DiHcRED moietymediated OCs.…”

Expanded spectrum of Pelizaeus–Merzbacher-like disease: literature revision and description of a novel GJC2 mutation in an unusually severe form

“…24 In addition, interactions between the aromatic rings of residues, potentially involving p-stacking, are known to have an important role in the self-assembly, by supplying energy, as well as in the correct orientation, through stacking geometries. 25,26 Notably, the molecular modelling of p.Ile33Met predicted that the replaced 33Met, being in short contact with the Trp3 side chain, could cause the displacement of the N-terminal helix of each Cx47 monomer towards the inner side of the hemichannel resulting in a potentially partial block of the pore. This 'in silico' prediction appears to be in line with the functional findings reported in 3 SPG44 patients by Orthmann-Murphy et al, 14 who observed-in a cell model system-that the mutant protein (I33M-Cx47) formed gap junction plaques that were indistinguishable from WT-Cx47, but the resulting I33M-Cx47/Cx43 channels showed severely altered voltage-dependent gating.…”