N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Abstract

The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.R eceptor tyrosine kinases (RTKs) are cell surface receptors that receive and transduce signals mediating a variety of critical cellular processes, including cell growth, migration, proliferation, differentiation, and apoptosis. Among the many RTKs, the most studied is the epidermal growth factor receptor (EGFR), not least because of its involvement in the development and progression of epidermoid cancers and its resulting importance as a target for antineoplastic therapies.Structurally, the EGFR consists of the ectodomain (ECD) (further subdivided into four subdomains, DI-IV), the transmembrane domain (TMD), and the intracellular tyrosine kinase domain (TKD). Ligand binding induces conformational transitions of the ECD that stabilize receptor dimerization, culminating in the activation of the intracellular TKD and subsequent propagation of the activation signal (1). To prevent receptor activation and signaling in the absence of ligand, the structurally tethered ECD of monomeric EGFR blocks the intrinsic capacity of the TMD and the intracellular TKD to dimerize (2). Ligand binding is believed to release the self-inhibitory tether and facilitate receptor oligomerization and activation (3-6). A detailed understanding of the structural regulation of the intact full-length receptors in their native membranes promises to reveal the molecular basis for receptor regulation (7); however, the methodological limitations associated with crystallizing transmembrane proteins, together with the high flexibility of the full-length receptor, have prevented high-resolution crystallographic analysis.To fill this gap, extensive atom-scale molecular dynamics (MD) simulations were recently performed to elucidate the structural dynamics of the EGFR in a two-component lipid bilayer (8). These studies suggest a large interfacial contact area between the membrane and the ecto-and intracellular domains of the unli...

Variation in Carbohydrates between Cancer and Normal Cell Membranes Revealed by Super‐Resolution Fluorescence Imaging

“…Additionally, changing density of reacting carbohydrate ligands may be accompanied with altering size and orientation of carbohydrates and the dimensional relationship between ligands and CBPs,25, 29 which will affect the accessibility of the carbohydrate ligands and finally promote or suppress the related processes. Moreover, alterations in protein glycosylations have been reported to perturb the structure and function of glycoproteins by changing their oligomerization, turnover, conformation, and interactions with other molecules 30, 31, 32, 33. Some computational models have also predicted that bulky glycoproteins favor transmembrane receptor organization (e.g., carbohydrate‐mediated integrin clustering would facilitate the assembly of mature adhesion complexes and enhance growth factor signaling) 34, 35.…”

Molecular electrometer and binding of cations to phospholipid bilayers

“…Furthermore, the overestimation of binding affinity may extend from ions to other positively charged objects, say, membrane protein seg-ments. This would affect lipid-protein interactions and could explain, for example, certain contradicting results on electrostatic interactions between charged protein segments and lipid bilayers 116,117 . In conclusion, more careful studies and model development on lipid bilayer-charged object interactions are urgently called for to make molecular dynamics simulations directly usable in a physiologically relevant electrolytic environment.…”

Moderate and strong static magnetic fields directly affect EGFR kinase domain orientation to inhibit cancer cell proliferation

“…In addition, SMFs could also affect lipid membrane composition and/or properties, which affect EGFR [40] and contribute to the cellular effects of SMFs on cells. The glycosylation of EGFR and the orientation of EGFR ectodomain relative to cell membrane [41], which are crucial for EGFR activity, may also be affected by SMFs.…”