STRUCTURAL CHARACTERIZATION OF A HOMOPHILIC BINDING-SITE IN THE NEURAL CELL-ADHESION MOLECULE

We have previously used synthetic peptides to identify a homophilic bi nding site between Lys-243 and Glu-252 (KYSFNYDGSE) in the third immun oglobulin-like domain of the chick neural cell adhesion molecule (NCAM ). In this report, we show that the deletion of this decapeptide seque nce from chick NCAM or the scrambling of the first 5 amino acid residu es led to the abolition of the homophilic binding activity of NCAM, th us confirming the role of this sequence in NCAM-NCAM binding. To inves tigate the involvement of individual residues of this decapeptide in N CAM binding, competition experiments were carried out using peptide an alogues with various amino acid substitutions. Substitution of both Ly s-243 and Asp-249 with Ala or of the 3 aromatic residues with Ala led to a total loss of activity, highlighting the importance of these resi dues in NCAM binding. Site-directed mutagenesis was then employed to s ubstitute individual amino acids within the decapeptide sequence with Ala. The homophilic binding activity of mutant NCAMs transiently expre ssed in COS-1 cells was determined using the NCAM-Covasphere binding a ssay. Substitution of the charged residues with alanine decreased NCAM binding activity, implicating electrostatic interactions in NCAM bind ing activity. Substitution of the aromatic residues Tyr-244 and Phe-24 6 with Ala abolished NCAM binding activity, suggesting that hydrophobi c and/or aromatic interactions may play an important role in NCAM homo philic binding. Substitution of amino acids in the predicted beta-stra nd portion of the decapeptide with Pro, which would tend to disrupt be ta-strand conformation, led to a substantial loss of activity. Thus, N CAM-NCAM binding may also depend on the beta-backbone structure of thi s site. These results are consistent with the involvement of multiple amino acids within the decapeptide sequence in NCAM homophilic interac tion.