Project Summary of the Funded Award (R01GM141298) To sense the environment, cells rely on membrane-embedded receptors. The receptor tyrosine kinase (RTK) family of signaling proteins is large, diverse, and centrally important both to human development diseases and cancers. Evidence so far supports a model that signal passage through RTKs is initiated by a structural change in the extracellular domain and then conducted through the transmembrane (TMD) and juxtamembrane (JMD) domains to the cytoplasmic kinase domain. The receptors usually are activated in the dimer form. Numerous RTK mutations confer diseases, e.g. single point mutations in ~30% of residues of the TMD of the fibroblast growth factor receptor 3 (FGFR3) are pathogenic, while mutations of tropomyosin receptor kinase A can lead to cancers. Understanding the structural interactions of the FGFR3 and TrkA signaling TMD and JMD therefore is crucial for fundamental biology and for future development of therapies that may target these pathways. Atomistically resolved TMD+JMD dimer structures are the major objective of this project. Application of traditional computational and crystallographic methods is hindered by the fluid nature of the membrane environment. Our goal is to develop novel efficient computational methods that guide and maximally leverage NMR, FRET, and in-cell experimental data and apply these methods to capture the FGFR3 and TrkA TMD and TMD+JMD dimer structures for the wild type and mutated pathogenic forms. In Aim 1, we will combine our novel highly mobile membrane mimetic model, capable of spontaneously capturing candidate TMD dimer structures, with a novel minimally biased way of applying a reduced number of computational restraints based on experimental distance measurements. The resulting TMD dimer structures will be validated by comparing computed and experimentally measured parameters. These structures will reveal the role mutations play in RTK dynamics. In Aim 2, we will use our computational-experimental approach to determine the role that juxtamembrane domains play in RTK signaling. The resolved structures of the mutated dimers will facilitate understanding of the pathology and mechanisms of receptor activation. Our novel computational approaches combined with extended expertise of co-investigators and collaborators in NMR, FRET, RTK signaling, and membrane-associated phenomena, uniquely position us to develop and apply this methodology. We will also develop an open-source, user friendly workflow plugin for a widely-used software suite that will allow efficient use of the proposed protocols by the scientific community. Completion of the specific aims will increase our ability to efficiently gain structural information on RTKs and will open new research avenues for investigating mechanisms of transmembrane signaling in health and disease leading to development of new treatments.

PROJECT NARRATIVE Disorders of cell signaling cascades are known to cause developmental pathologies, inflammation, diabetes, and cancers, while the mechanisms of signaling in health and disease are not well understood. Transmembrane and juxtamembrane domains of the structurally diverse receptor tyrosine kinases are central to the signal passage into the cell and numerous point mutations of these domains are attributed to diseases, yet structures of their dimers are lacking. Our goals are to develop a versatile method that combines novel computational approaches with experimental data, and to capture long-overdue dynamic dimer structures of the wild type and the pathogenic mutated forms of representative receptor tyrosine kinase subfamilies, improving our understanding of the mechanisms of cell signaling in disease and leading to new treatments.