Structural and Functional Characterization of Alternative Transmembrane Domain Conformations in VEGF Receptor 2 Activation

Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy. •The extracellular domain prevents ligand-independent VEGFR-2 activation•Specific transmembrane helix orientation is required for VEGFR-2 activation•The structure of the transmembrane domain of VEGFR-2 was analyzed by NMR spectroscopy•Molecular dynamics simulations confirm the conclusions drawn from NMR structures Manni et al. describe the role of the VEGF receptor transmembrane domain in receptor signaling. The authors correlate biochemical analyses on mutant receptors carrying dimerization-promoting transmembrane domains with structural data obtained by NMR spectroscopy and molecular dynamics simulation.