Conformational equilibria and intrinsic affinities define integrin activation.

Jing Li,Timothy A Springer,Carlos Cabañas,Wei Xia,Chafen Lu,Martin J Humphries,Yang Su,Dietmar Vestweber,Yan Qin

doi:10.15252/embj.201695803

Abstract

We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000‐fold higher affinity of the extended‐open state than the bent‐closed and extended‐closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1. On the surface of K562 cells, α5β1 is 99.8% bent‐closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N‐glycans and leg domains in each subunit that connect the ligand‐binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single‐span transmembrane domains.

Highlights

To quantitatively relate the steps involved in signal transmission across the plasma membrane in cell surface receptors, an understanding of receptor energy landscapes is essential
Principles for saturable stabilization of defined conformational states Here, we describe the fluorescence polarization (FP) assay used in much of this work, and the methods required to establish that the measurements reflect values for the desired conformational states
Using fixed concentrations of integrin and fluorescein isothiocyanate (FITC)-labeled cyclic RGD peptide (Koivunen et al, 1995; Xia & Springer, 2014) and titrating in Fabs, we measured the ability of Fabs to increase or decrease ligand binding by altering integrin affinity for cRGD

Summary

Introduction

To quantitatively relate the steps involved in signal transmission across the plasma membrane in cell surface receptors, an understanding of receptor energy landscapes is essential. The gaps in free energy between signal-competent and incompetent receptor conformational states are especially important. Such understanding is currently limited to receptors with large lipid-embedded domains, such as G protein-coupled receptors and ion channels (Ruiz & Karpen, 1997; Horrigan et al, 1999; Lape et al, 2008; Park et al, 2008; Cecchini & Changeux, 2015; Manglik et al, 2015). Much less is known about receptors that bind ligands through extracellular domains that are in tandem with single-span transmembrane domains, including integrins. The b-propeller domain in a and the bI domain in b associate to form a ligandbinding head, which is linked to leg, transmembrane, and cytoplasmic domains in each subunit

Methods

Results

Conclusion