[F31] Integrating Structural and Biochemical Approaches to Define Lipid-Dependent TAM Receptor Activation

Ente: National Institute of Allergy and Infectious Diseases

Scadenza: 2029-08-23

Importo max: 41.340 EUR

Paese: US

Descrizione

ABSTRACT TAM receptors (Tyro3, Axl, and Mer) are receptor tyrosine kinases that couple apoptotic cell clearance to the regulation of inflammation and immune homeostasis, and their dysregulation contributes to autoimmune disease, neurodegeneration, impaired pathogen clearance, infertility, and cancer. Tyro3 is the least understood TAM receptor, yet has been associated with asthma susceptibility, neurodegeneration, and cancer progression, making it an emerging therapeutic target. TAM receptors are activated by the vitamin K–dependent ligands Gas6 and Protein S, which bind phosphatidylserine (PtdSer) on apoptotic cells and bridge to the extracellular Ig domains of the receptors, assembling a lipid–ligand–receptor signaling complex. Although the Axl:Gas6 complex has been structurally characterized, the mechanistic basis of Tyro3 and Mer activation—particularly how they recognize Gas6 versus Protein S and how PtdSer remodels these assemblies—remains unknown. Preliminary data from our lab show that Tyro3 forms ligand-independent dimers, displays distinct preferences for Gas6 and Protein S, and requires PtdSer for full activation in a manner that differs from both Axl and Mer. I therefore hypothesize that Tyro3 activation is governed by unique structural mechanisms shaped by ligand-specific contacts and lipid-dependent remodeling of the ligand–receptor assembly, and that these mechanisms differ fundamentally from those of Axl and Mer. To test this, I will integrate structural modeling, cell-based signaling assays, quantitative biophysics, X-ray crystallography, cryo-electron microscopy (cryo-EM), and lipid nanodisc reconstitution across three Aims. In Aim 1, I will define residue-level interaction sites that govern Tyro3 and Mer recognition of Gas6 and Protein S by testing a focused panel of 36 receptor mutants in phosphorylation assays in TAM-TKO cells and in surface plasmon resonance binding experiments. These studies will identify the key extracellular determinants that couple ligand and lipid engagement to downstream signaling. In Aim 2, I will determine the structural basis of Tyro3 activation by solving high-resolution structures of the Tyro3 extracellular domain in its unbound (apo) state by X-ray crystallography and in complex with full-length Gas6 by single-particle cryo-EM, revealing how ligand binding reorganizes pre-assembled dimers into activation-competent conformations. In Aim 3, I will determine how PtdSer remodels TAM assemblies using Axl:Gas6 reconstituted into PtdSer-containing nanodiscs as a benchmark lipid-dependent system, comparing nanodisc-engaged and lipid-free complexes to define PtdSer- driven domain rearrangements and clustering principles. Together, these studies will establish the first integrated structural and mechanistic framework for Tyro3 activation, explain how ligand identity and lipid engagement tune TAM signaling, and lay the groundwork for rational design of Tyro3-directed therapeutics in cancer and inflammatory d Istituzione: UNIV OF NORTH CAROLINA CHAPEL HILL PI: Maria Camila Arango Progetto: 1F31AI203078-01

Settori: National Institute of Allergy and Infectious Diseases

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