A Conserved Ectodomain-Transmembrane Domain Linker Motif Tunes the Allosteric Regulation of Cell Surface Receptors

Abstract

In many families of cell surface receptors, a single transmembrane (TM) ␣-helix separates ecto-and cytosolic domains. A defined coupling of ecto-and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin ␣IIb in model constructs and relate it to other integrin ␣ subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit ␤ and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors.Cells sense their environment through transmembrane (TM) 3 surface receptors that transmit extracellular signals into the cell. With the exception of G-protein coupled receptors, these proteins are dimers composed of subunits that each typically exhibits an extracellular domain (ectodomain) containing the ligand-binding site, a single TM ␣-helix, and an intracellular effector domain. Integrins, which control vital cell-cell and cellmatrix adhesions (1-3), constitute one ubiquitous family of TM cell surface receptors. Receptor-tyrosine kinases represent another prominent family that activate upon the stabilization of dimeric receptor states by bound ligand (4, 5). However, rather than being simple binary switches, all these receptors have the potential for allosteric regulation (1, 5, 6), indicating a multistate coupling of ecto-and TM domains. This coupling and associated allosteric parameters must depend on the structural and dynamic properties of the linker between ecto-and TM domains. However, irrespective of detailed structural information on ecto-and TM domain in receptor-tyrosine kinases (4, 5), structural or dynamic information on linkers remains indirect and ambiguous (7-9). Similarly, structures of integrin ectodomains, TM domains, and cytosolic tails are available (10 -16) but little is known about their ecto-TM domain coupling. In support of a functionally relevant linker, integrin ␣M␤2 spontaneously activates when the TM domain is uncoupled from the ectodomain by insertion of a flexible (GGGGS) 2 linker (17).Integrins consist of heterodimeric, non-covalently associated ␣␤ subunits that each exhibits a large ectodomain, a single TM ␣-helix, and a short cytosolic tail (Fig. 1A). In addition to outside-in signaling, integrins also signal in the opposite direction (inside-out signaling) in relationship to their function as dynamic c...

Energy landscape differences among integrins establish the framework for understanding activation

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“…Black bars indicate the TMD borders as reported in ref. 45 . ( c ) Two dimensional histograms of the RMSD values of all C α atoms (ordinate values) and only those that are embedded in the membrane (abscissa values) (range of residues considered: P996-V1015 and D718-I747) calculated over three MD simulations.…”

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