ABSTRACT
Common γ chain (γc) cytokine receptors, including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with a common γc receptor (CD132) by concomitant binding of their ectodomains to an interleukin. In this work, we find that direct interactions between the transmembrane domains (TMDs) of both the γc and the interleukin receptors (ILRs) are also required for receptor activation. Moreover, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences within the family. Heterodimer structures of γc TMD bound to IL-7 and IL-9 receptor TMDs-determined in a lipid bilayer-like environment by nuclear magnetic resonance spectroscopy-reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Thus, signaling in the γc receptor family requires specific heterotypic interactions of the TMDs.
Subject(s)
Interleukin Receptor Common gamma Subunit , Interleukin-7 Receptor alpha Subunit , Protein Interaction Domains and Motifs , Interleukin Receptor Common gamma Subunit/chemistry , Interleukin Receptor Common gamma Subunit/genetics , Protein Binding , Signal Transduction , Nuclear Magnetic Resonance, Biomolecular , Interleukin-7 Receptor alpha Subunit/chemistry , Interleukin-7 Receptor alpha Subunit/geneticsABSTRACT
The common γ-chain (γc) family of cytokine receptors, including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with the common γc receptor in ligand dependent manner. Sharing of γc by the IL receptors (ILRs) is thought to be achieved by concomitant binding of γc and ILR ectodomains to a cytokine. Here, we found that direct interactions between the transmembrane domain (TMD) of γc and those of the ILRs are also required for receptor activation, and remarkably, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences. Heterodimer structures of γc TMD bound to the TMDs of IL-7R and IL-9R, determined in near lipid bilayer environment, reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Functional mutagenesis data indicate the requirement of the heterotypic interactions of TMDs in signaling, which could explain disease mutations within the receptor TMDs. One-Sentence Summary: The transmembrane anchors of interleukin receptors of the gamma-chain family are critical for receptor sharing and activation.
ABSTRACT
The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows for significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins. The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodisks, and surfactants to allow for the expression of toxic membrane proteins or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase, and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high-molecular-weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.
