Cellular Footprinting of Transferrin-Receptor Complex

Case Western Reserve University, Cleveland OH

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): The molecular mechanisms of ligand binding to membrane receptors are important to a variety of cell functions including hormone action, immune responses, metabolite transport, and nerve transmission. Although significant advances have been made in determining membrane protein structures using crystallography, NMR, and cryo-electron microscopy, methods to examine the details of membrane protein structure and dynamics in situ are few. In this R21 proposal under PA-02-008, we will establish the feasibility of a cellular protein footprinting assay to provide molecular details of the conformational changes of transferrin when it binds its membrane bound receptor. In Aim 1, we will develop biochemical and mass spectrometry pull-down methods to isolate HIS6- tagged-transferrin specifically bound to transferrin receptors in K562 cells. We will optimize protease digestion and LC-MS methods to examine transferrin peptides derived from the pull-downs. In Aim 2 we will determine appropriate conditions for synchrotron footprinting in the presence of K562 cells including defining appropriate dose and buffer conditions for footprinting. Lastly, in Aim 3, we will determine the molecular details of the conformational changes that occur in transferrin when it binds its membrane bound receptor in a K562 cell based footprinting assay and compare the structural changes to in vitro footprinting and cryo-EM data on transferrin binding to the recombinant receptor ectodomain, which lacks the membrane attachment "stalk". These studies, if successful, will provide specific cell based tests of the predicted side-chain interactions provided by cryo-EM, allow comparison to in vitro footprinting results, and most importantly, demonstrate a novel method of structural analysis for examining membrane protein structure, dynamics and ligand binding in a live cell assay.