Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices

J Biol Chem. 1992 Apr 15;267(11):7683-9.

Abstract

Specific side-by-side interactions between transmembrane alpha-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane alpha-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane alpha-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the alpha-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cell Membrane / metabolism
  • Circular Dichroism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Glycophorins / genetics
  • Glycophorins / metabolism*
  • Humans
  • Micrococcal Nuclease / genetics
  • Micrococcal Nuclease / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Glycophorins
  • Recombinant Fusion Proteins
  • Micrococcal Nuclease