Effects of the Oncogenic V_(664)E Mutation on Membrane Insertion, Structure, and Sequence-Dependent Interactions of the Neu Transmembrane Domain in Micelles and Model Membranes: An Integrated Biophysical and Simulation Study

摘要

Receptor tyrosine kinases bind ligands such as cytokines, hormones, and growth factors and regulate key cellular processes, including cell division. They are also implicated in the development of many types of cancer. One such example is the Neu receptor tyrosine kinase found in rats (homologous to the human ErbB2 protein), which can undergo a valine to glutamic acid (V_(664)E) mutation at the center of its a-helical transmembrane domain. This substitution results in receptor activation and oncogenesis. The molecular basis of this dramatic change m behavior upon introduction of the V_(664)Emutation has been difficult to pin down, with conflicting results reported in the literature. Here we report the first quantitative, thermodynamic analysis of dimerization and biophysical characterization of the rat Neu transmembrane domain and several mutants in a range of chemical environments. These data have allowed us to identify the effects of the V_(664)E mutation in the isolated TM domain with respect to protein-protein and protein-lipid interactions, membrane insertion, and secondary structure. We also report the results from a 100 ns atomistic molecular dynamics simulation of the Neu transmembrane domain in a model membrane bilayer (dipalmitoylphosphatidylcholine). The results from simulation and experiment are in close agreement and suggest that, in the model systems investigated, the V_(664)E mutation leads to a weakening of the TM dimer and a change in sequence-dependent interactions. These results are contrary to recent results obtained in mammalian membranes, and the implications of this are discussed.