Val(659)-> Glu mutation within the transmembrane domain of ErbB-2: Effectsmeasured by H-2 NMR in fluid phospholipid bilayers

Certain point mutations within the hydrophobic transmembrane domains of cla ss I receptor tyrosine kinases have been associated with oncogenic transfor mation 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 t he oncogenic nature of this Val-->Glu substitution may derive from alterati on of the transmembrane domain's ability to take part in direct side-to-sid e associations. In the present work, we examined the basis of this phenomen on by studying transmembrane portions of ErbB-2 in fluid bilayer membranes. An expression system was designed to produce such peptides from the wild-t ype ErbB-2, and from an identical region of the transforming mutant in whic h Val(659) is replaced by Glu. All peptides were 50-mers, containing the ap propriate transmembrane domain plus contiguous stretches of amino acids fro m the cytoplasmic and extracellular domains. Deuterium heteronuclear probes were incorporated into alanine side chains (thus, each alanine -CH3 side c hain became -CD3). Given the presence of natural alanine residues at positi ons 648 and 657 within ErbB-2, this approach afforded heteronuclear probes within the motif Ser(656)AlaValValGlu(660), thought to be important for hom odimer formation, and nine residues upstream of this site. Further peptides were produced, by site-directed mutagenesis, to confirm spectral assignmen ts and to provide an additional probe location at position 670 (11 residues downstream of the motif region). On SDS-polyacrylamide gels, the transmemb rane peptides migrated as predominant monomers in equilibrium with smaller populations of homodimers/oligomers. CD spectra of both wild-type and trans forming mutant peptides were consistent with the transmembrane portions bei ng basically alpha-helical. H-2 NMR spectra of each transmembrane peptide w ere obtained in fluid phospholipid bilayers of 1-palmitoyl-2-oleoylphosphat idylcholine (POPC) from 35 to 65 degrees C. Results were consistent with th e concept that the glutamic acid residue characterizing the mutant is uncha rged at neutral pH. Narrowed spectral components from species rotating rapi dly and symmetrically within the membrane appeared to represent monomeric p eptide. Mutation of Val(659) to Glu within the hydrophobic domain induced c hanges 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 format ion), 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 re sult argues that the transforming mutation is associated with significant i ntramolecular 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 suggeste d that there is greater peptide backbone flexibility for the wild-type tran smembrane region.