Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

Abstract

SUMMARY Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region, but cannot explain the ‘high-affinity’ and ‘low-affinity’ classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors, and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

A single ligand is sufficient to activate EGFR dimers

“…2 and SI Appendix, Fig. S4, and Table S3) (11,13,15,16). The C-terminal juxtamembrane regions of domain IV are closely apposed when well ordered, suggesting interactions between transmembrane regions in active receptor dimers, but regions of domain IV homologous to those involved in direct dimer contacts in EGFR:EGF complexes (13) are mostly disordered and few specific intersubunit domain IV contacts are resolvable.…”

“…At odds with a ligand-induced dimerization model of EGFR signaling, however, are recent studies showing dimers of human EGFR in the absence of ligand (5-8) as well as negative cooperativity when epidermal growth factor (EGF) binds to EGFR (9). Curiously, the single Drosophila EGFR homolog adopts an extended conformation in the absence of ligand and forms asymmetric receptor dimers with a single high-affinity ligand bound (10,11), suggesting different mechanisms may regulate EGFR activation in Drosophila and humans.…”

Asymmetric perturbations of signalling oligomers

“…By monitoring the fluorescence of the ligand or that of the target protein, fluorescence spectroscopy is well suited to reveal phenomena such as binding cooperativity (Alvarado, 2010) and by the application of special labelling techniques, distinguish between cis and trans activation in GPCR dimers (Damian et al, 2006(Damian et al, , 2008. Site-directed fluorescence spectroscopy can also be used to identify different structural domains associated with asymmetric ligand binding (Sommer et al, 2012).…”

“…The XRC of EC and IC domains revealed that i) the C-lobe of the donor kinase domain asymmetrically interacts with the N-lobe of the acceptor domain (Zhang et al, 2006) and ii) an IC juxtamembrane segment (latch) is crucial for dimerization of the kinase domains and activation (Jura et al, 2009). Fluorescent ligand-binding assays demonstrated that the first EGF binding promotes an asymmetric dimerization interface (Alvarado et al, 2010). As a result of EGF binding to a luciferase-labelled heterodimer the cis-kinase adopted the acceptor position in the asymmetric dimer and was activated first (Macdonald-Obermann et al, 2012).…”

“…X-ray crystallography X-ray crystallography (XRC) has become the major method for high-resolution structure elucidation of proteins. Due to the obstacles of crystallization, the XRC structures of just the extracellular (EC) ligand-binding or intracellular (IC) functional coupling domains have been determined at first for several membrane-bound signalling proteins (Alvarado et al, 2010;Kunishima et al, 2000). To overcome the difficulties posed by the relatively small polar surface of membrane proteins, shorter-chain detergents in combination with amphiphile additives, less hydrophobic point mutations, protein fusion, complexation with an antibody fragment or with a crystallization 'chaperone' have been introduced into conventional mixed micelle crystallization techniques (Caffrey et al, 2012).…”