Val(659)-->Glu mutation within the transmembrane domain of ErbB-2: effects measured by (2)H NMR in fluid phospholipid bilayers.
Certain point mutations within the hydrophobic transmembrane domains of class I receptor tyrosine kinases have been associated with oncogenic transformation in vitro and in vivo [Gullick, J., and Srinivasan, R. (1998) Breast Cancer Res. Treat. 52, 43-53]. An important example is the replacement of a single (hydrophobic) valine by (charged) glutamate in the rat protein, Neu, and in the homologous human protein, ErbB-2. It has been suggested that the oncogenic nature of this Val-->Glu substitution may derive from alteration of the transmembrane domain's ability to take part in direct side-to-side associations. In the present work, we examined the basis of this phenomenon by studying transmembrane portions of ErbB-2 in fluid bilayer membranes. An expression system was designed to produce such peptides from the wild-type ErbB-2, and from an identical region of the transforming mutant in which Val(659) is replaced by Glu. All peptides were 50-mers, containing the appropriate transmembrane domain plus contiguous stretches of amino acids from the cytoplasmic and extracellular domains. Deuterium heteronuclear probes were incorporated into alanine side chains (thus, each alanine -CH(3) side chain became -CD(3)). Given the presence of natural alanine residues at positions 648 and 657 within ErbB-2, this approach afforded heteronuclear probes within the motif Ser(656)AlaValValGlu(660), thought to be important for homodimer formation, and nine residues upstream of this site. Further peptides were produced, by site-directed mutagenesis, to confirm spectral assignments and to provide an additional probe location at position 670 (11 residues downstream of the motif region). On SDS-polyacrylamide gels, the transmembrane peptides migrated as predominant monomers in equilibrium with smaller populations of homodimers/-oligomers. CD spectra of both wild-type and transforming mutant peptides were consistent with the transmembrane portions being basically alpha-helical. (2)H NMR spectra of each transmembrane peptide were obtained in fluid phospholipid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) from 35 to 65 degrees C. Results were consistent with the concept that the glutamic acid residue characterizing the mutant is uncharged at neutral pH. Narrowed spectral components from species rotating rapidly and symmetrically within the membrane appeared to represent monomeric peptide. Mutation of Val(659) to Glu within the hydrophobic domain induced changes in side chain angulation of at least 6-8 degrees at Ala(657) (i.e., within the five amino acid motif thought to be involved in homodimer formation), and downstream of this site to residue 670. There was little evidence of effect at the upstream site (Ala(648)) at the membrane surface. This result argues that the transforming mutation is associated with significant intramolecular rearrangement of the monomeric transmembrane helix-extending over some four helix turns-which could influence its lateral associations. In addition, temperature effects on spectral quadrupole splittings suggested that there is greater peptide backbone flexibility for the wild-type transmembrane region.
Sharpe
S
S
Department of Biochemistry, University of Western Ontario, London, Canada.
Barber
K R
KR
Grant
C W
CW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D003903:Deuterium; D019281:Dimerization; D018734:Genes, erbB-2; D005998:Glycine; D006801:Humans; D008051:Lipid Bilayers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010713:Phosphatidylcholines; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014633:Valine
D003903:Deuterium; D005998:Glycine; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D014633:Valine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine