Structural determination of TrkB assembly and activation - PROJECT SUMMARY The proposed project encompasses a comprehensive research plan to support the development of my career as an independent scientist focused on investigating the assembly and conformational dynamics of neuronal integral membrane receptors, and how structure is related to receptor activation and regulation. This biophysical mechanism-focused approach to receptor function will guide promising therapeutic development. Background: The neurotrophic receptor tropomyosin kinase receptor B (TrkB) is a member of the receptor tyrosine kinase (RTK) superfamily of integral membrane proteins which receive extracellular signals and initiate intracellular signaling cascades across the cellular membrane. Canonical RTK activation is characterized by a transition from a monomeric receptor to a dimeric receptor following ligand binding. However, receptor assembly studies on EGFR, an extensively studied RTK, demonstrate that this activation pathway is much more complex than initially thought as an ensemble of ligand-dependent conformations have been observed. Despite the role that TrkB plays in neurodegenerative, psychiatric, and oncological disease, no effective therapeutic agent that targets TrkB is available due in part to the overwhelming lack of structural information relating to receptor function. Specifically, the field currently lacks an understanding of TrkB dimeric assembly and how this relates to intersubunit and intrasubunit conformational dynamics across domains. Specific Aims and Research Design: The proposed study investigates TrkB assembly, structure, and conformational dyanmics using 1) single-molecule and ensemble fluorescence-based binding methods to investigate the molecular determinants of TrkB dimerization 2) cryogenic electron microscopy (cryoEM) to assess the structural basis of TrkB dimeric assembly and allosteric activation. My in vitro and structural findings will be validated and extended by cellular and functional assays. Together this work will guide the development of a K99/R00 proposal aimed to apply the approaches and findings from this F32 research program broadly to other neuronal integral membrane receptors and to pursue the functional consequences of TrkB receptor assembly and conformational shifts in in vivo models. Training and Mentoring: My training goals are supported by 1) my sponsor Dr. Levitz and my assembled team of carefully selected consultants and collaborators with a range of expertise in membrane protein biophysical and biochemical research, 2) a flourishing institutional environment at Weill Cornell Medical College, and 3) scientific meetings, seminars, and planned publications. Impact: The invaluable experience gained during this award will serve as the foundation for my independent career as a protein biophysics researcher and for the development of a novel program of membrane protein research.
