title: Beta-branched residues adjacent to GG4 motifs promote the efficient association of glycophorin A transmembrane helices.
abstract: Protein transmenembrane (TM) segments participating in helix-helix packing commonly contain small residue patterns (termed GG4 or "small-xxx-small" motifs) at i and i + 4 positions. Within many TM segments - such as the glycophorin A (GpA) sequence L75IxxGVxxGVxxT87- the G17y-xxx-Gly83 motif often occurs in combination with large, usually beta3-branched aliphatic residues at adjacent positions, typified here by Val30 and Val84 residues. To explore the importance of local P-branched character on GpA dimerization, we made systematic replacements to all 16 combinations of single or double Ile, Leu, and AIa residues at GpA TM Val/Val positions 80 and 84. Using the TOXCAT system to assay self-oligomerization in the Escherichia coli inner membrane--we observed that (i) combinations of Val and lie residues maintained, or improved dimerization levels; (ii) single Ala or Leu mutant combinations with Val or Ile maintained near-wild type dimerization affinities; and (iii) in the absence of beta-branching, i.e., Leu/Leu, Ala/Ala and Ala/Leu combinations, GpA dimerization was significantly diminished. An apparent capacity of lle-containing mutants to increase GpA dimerization versus WT likely arises from improved van der Waals packing (vs. Val) within the locus of helix contact, consistent with correlations we noted in lipid accessibility measurements. Examination of several synthetic peptides with sequences corresponding to selected GpA mutants (VV VI, IV II, and LL) confirmed their dimerization on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The overall results reinforce the importance of a beta-branch-containing "ridge" residue to complement a "small-xxx-small groove" in promotion of TM-TM interactions.
authors.affiliation: Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Fiona
authors.initials: F
authors.lastname: Cunningham
authors.forename: Bradley E
authors.initials: BE
authors.lastname: Poulsen
authors.forename: Wilfred
authors.initials: W
authors.lastname: Ip
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D020816:Amino Acid Motifs; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006021:Glycophorins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D055503:Protein Multimerization / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006021:Glycophorins; D011994:Recombinant Proteins