Efficient sampling of TrkA transmembrane domain dimerization captures functionally distinct structural ensembles

Upon ligand binding, the transmembrane domains (TMD) of the receptor tyrosine kinase (RTK) dimerize, induce conformational change in the juxtamembrane domain (JMD), and trigger downstream phosphorylation that eventually determines fate of the cell. To overcome the experimental difficulty in probing the TMD dimerization events in membrane bilayers, we employed all-atom molecular dynamics with a novel membrane mimetic solvent Simple Carbon Solvent Ethane (SCSE) to efficiently sample the TMD dimerization processes for tropomyosin receptor kinase A (TrkA). Spontaneous and stable TMD dimers were observed in less than 50 ns of simulation time from an initially separated grid configuration. We then performed hierarchical clustering using a customized distance metric based on TMD contact residues, and revealed major structural ensembles of TMD dimers. We demonstrated the validity of the resolved TMD dimer structures by performing MM-GBSA free energy calculation and molecular dynamics with full-tail lipid bilayers. As the residues in contact in the most populated TMD dimer ensemble from our SCSE simulations overlap with those found to be critical for kinase activation, we hypothesized that the captured TMD dimer corresponds to the active state, while the previously characterized NMR structure is the inactive state. To understand the role of different TMD dimer conformations in downstream signaling, we assembled TMD+JMD constructs using the captured in simulation dimer structure and the NMR structure. We modelled the system with Highly Mobile Membrane Mimetic (HMMM) for accelerated peptide-lipid interactions and found that the captured TMD dimer leads to JMD conformations distinct from those tethered to the NMR dimer structure, which may enable the kinase domain for cross-phosphorylation. Our method represents a new approach to study RTK dimerization and sample physiologically relevant dimer structures.