Structural Basis of the Differential Stability and Receptor Specificity of H-2Db in Complex with Murine versus Human β2-Microglobulin

β2-Microglobulin (β2m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human β2m (hβ2m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse β2m (mβ2m) with hβ2m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific β2m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2Db in complex with LCMV-derived gp33 peptide and either hβ2m or mβ2m. The conformation of the gp33 peptide is not affected by the β2m species. Comparison of the interface between β2m and the α1α2 domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hβ2m and the α1α2 domains of H-2Db. We propose that the positioning of two hydrogen bond rich regions at the hβ2m/α1α2 interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2Db/hβ2m complex. These two regions act as bridges, holding and stabilizing the underside of the α1 and α2 helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2Db in complex with either mβ2m or hβ2m provides a structural explanation for the differential binding of H-2Db/hβ2m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of β2m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hβ2m impairs the recognition of Ly49C by H-2Db/gp33/hβ2m. Finally, comparison of the two H-2Db crystal structures implies that the β2m species may affect the strength of TCR recognition by affecting CD8 binding.