Deviant trafficking of I-A(d) mutant molecules is reflected in their peptide binding properties

I-A(d) molecules harboring single amino acid changes in the conserved 80-82 region of the beta-chain show altered trafficking in invariant chain (li)- negative cell lines. Since residues beta 81 and beta 82 form hydrogen bonds with the backbone of bound peptide, alterations in this region may result in distinct MHC class II conformers that are targeted aberrantly. We examin ed the assembly and peptide binding properties of the mutant I-A(d) molecul es generated by in vitro translation. Indeed, loss of a single hydrogen bon d at beta 81, or of two hydrogen bonds at beta 82, is sufficient to render I-A(d) incapable of stable interaction with CLIP and other antigenic peptid es, despite normal assembly with intact invariant chain. These results sugg est that stable interaction of MHC class II molecules with peptide requires the integrity of the H-bond network between residues in the MHC class II a lpha-helices and bound peptide, and that conformational features revealed b y stable peptide binding are critical for MHC class II intracellular transp ort.