The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.

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.