The Effect of Hydrophilic Substitutions and Anionic Lipids upon the Transverse Positioning of the Transmembrane Helix of the ErbB2 (neu) Protein Incorporated into Model Membrane Vesicles

Abstract

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H>E>Q>K>G>V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.