Motifs of Two Small Residues can Assist but are not Sufficient to Mediate Transmembrane Helix Interactions

Both experimental and statistical searches for specific motifs that mediate transmembrane helix–helix interactions showed that two glycine residues separated by three intervening residues (GxxxG) provide a framework for specific interactions. Further work suggested that other motifs of small residues can mediate the interaction of transmembrane domains, so that the AxxxA-motif could also drive strong interactions of α-helices in soluble proteins. Thus, all these data indicate that a motif of two small residues in a distance of four might be enough to provide a framework for transmembrane helix–helix interaction. To test whether GxxxG is equivalent to (small)xxx(small), we investigated the effect of a substitution of either of the two Gly residues in the glycophorin A GxxxG-motif by Ala or Ser using the recently developed GALLEX system. The results of this mutational study demonstrate that, while a replacement of either of the two Gly by Ala strongly disrupts GpA homo-dimerization, the mutation to Ser partly stabilizes a dimeric structure. We suggest that the Ser residue can form a hydrogen bond with a backbone carbonyl group of the adjacent helix stabilizing a preformed homo-dimer. While (small)xxx(small) serves as a useful clue, the context of adjacent side-chains is essential for stable helix interaction, so each case must be tested.