Molecular and functional interaction of single pass ephrin receptor tyrosine kinase, EphA2 in lipid nanodisc using solution NMR.

Abstract

Eph receptor, the largest subfamily of single pass trans membrane receptor tyrosine kinase family play crucial role as a regulator during embryonic development, cell maturation, and adulthood. EphA2, a member of the Eph family receptor tyrosine kinases (RTK) is a key regulator of tumorigenesis and cancer progression. EphA2 can be activated canonically by ephrin ligand which normally is a repulsive signal OR non-canonical (unliganded EphA2) promoting cell migration in several cancer types. Here we focus to dissect the effects of the sterile α motif (SAM) domain together with the phosphorylation on intracellular region (ICR) interactions as well as the transmembrane construct of EphA2 in solution to understand the signaling mechanism at molecular level. Our study of the ICR shows that the sterile α motif (SAM) domain acts as a regulator impeding kinase domains interaction and conversely increasing the kinase activity upon deletion of SAM domain. While the presence of the SAM domain hinders oligomerization of phosphorylated ICR, it appears that such protein-protein interactions are required for kinase activity regulation. Mutation studies of the kinase domain, SAM domain and the linker region between kinase domain and SAM domain give insight into its regulatory role for EphA2 activity. More importantly, we have recently optimized truncated construct with single Fibronectin-III, FN-III (Ecto)-Transmembrane (TM)-Juxta-Membrane, JM (ICR) in presence of DDM with well dispersed peaks in TROSY-HQSC indicating well folded protein. We will incorporate this truncated construct into MSP lipid nanodisc for NMR studies and biophysical characterization. In addition to the ICR regulation via SAM domain, we will have in-depth perspective on the configurational coupling of membrane-proximal transmembrane domains to each other.