Distinct Conformations of Ly49 Natural Killer Cell Receptors Mediate MHC Class I Recognition in Trans and Cis

Abstract

Summary Certain cell surface receptors engage ligands expressed on juxtaposed cells, but also ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we show that Ly49 natural killer (NK) cell receptors bind two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains are back-folded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allow monovalent binding of MHC-I in cis. The distinct conformations (back-folded and extended) define the structural basis for cis–trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans, but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.

A Positive Cooperativity Binding Model between Ly49 Natural Killer Cell Receptors and the Viral Immunoevasin m157

“…The protein was produced as described for Ly49L (21). Briefly, Ly49H was expressed in Escherichia coli BL21(DE3) as inclusion bodies; solubilized in 6 M guanidine; and folded in vitro by dilution into 30% (v/v) glycerol, 0.4 M arginine, 50 mM Hepes (pH 7.0), 100 mM NaCl, 3 mM reduced glutathione, and 0.8 mM oxidized glutathione.…”

“…Considerable structural information is available for Ly49 receptors in MHC-bound and unbound forms. Crystal structures have been reported for Ly49A NKD in complex with H-2D d (16,17), Ly49C NKD bound to H-2K b (18,19), Ly49C NKD (19), Ly49I NKD (20), Ly49G2 NKD (19), Ly49L NKD (21), and Ly49L NKD with the stalk region (21). Collectively, these studies have shown that Ly49 receptors can adopt two distinct conformations: 1) a backfolded conformation in which the NKDs of the Ly49 dimer are backfolded onto the helical stalk region (Fig.…”

“…1B). These two conformations mediate MHC-I recognition in trans and cis (21). The backfolded conformation enables Ly49 to engage two MHC-I molecules in trans (Ly49 on the NK cell and MHC-I on a juxtaposed target cell) (Fig.…”

“…1D). Moreover, Ly49 receptors appear able to switch between backfolded and extended conformations (21).…”

Optimized Tetramer Analysis Reveals Ly49 Promiscuity for MHC Ligands

“…This is referred to as cis interaction, whereas binding to MHC-I on another cell is a trans interaction. Cis and trans interactions have two distinct receptor conformations made possible due to the flexible stalk; the stalk is able to bend into a back-fold conformation allowing for trans binding and straightens into an extended conformation for cis interactions (18). Cis interactions are stable and sequester Ly49A so that most receptors are not available for MHC-I binding in trans (19).…”