Mapping the major interaction between binding protein and Ig light chains to sites within the variable domain

Newly synthesized Ig chains are known to interact in vivo with the binding protein (BiP). a major peptide-binding chaperone in the endoplasmic reticul um, The predominant interactions between the light chain and BiP are observ ed early in the folding pathway, when the light chain is either completely reduced, or has only one disulfide bond. In this study, we describe the in vitro reconstitution of BiP binding to the variable domain of light chains (V-L). Binding of deliberately unfolded V-L was dramatically more avid than that of folded V-L, mimicking the interaction in vivo. Furthermore, V-L bi nding was inhibited by addition of ATP, was competed with excess unlabeled V-L, and was demonstrated with several different V-L proteins. Using this a ssay, peptides derived from the V-L sequence were tested experimentally for their ability to bind BiP. Four peptides from both beta sheets of V-L were shown to bind BiP specifically, two with significantly higher affinity. As few as these two peptide sites, one from each beta sheet of V-L, are suffi cient to explain the association of BiP with the entire light chain. These results suggest how BiP directs the folding of Ig in vivo and how it may be used in shaping the B cell repertoire.