IDENTIFICATION OF RESIDUES ON CD40 AND ITS LIGAND WHICH ARE CRITICAL FOR THE RECEPTOR-LIGAND INTERACTION

Interactions between gp39 (CD40L, TRAP, T-BAM) on activated T cells an d CD40 on antigen-presenting cells play an important role in regulatin g antibody production by B cells, cytokine production by monocytes, an d other immune responses which require T cell ''help''. Using structur e-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD 40 and gp39 surface residues which are important for receptor-ligand b inding. Binding studies with CD40 or gp39 proteins containing single a nd double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y1 45 are important for CD40 binding. Analysis of the location of amino a cid substitutions in the naturally occurring gp39 mutants expressed by the X-linked hyper-IgM (X-HIM) patients studied to date indicated the E129/G substitution found in the S128/R-E129/G double mutant affects a solvent-accessible residue which might participate in CD40/gp39 bind ing. Binding studies with E129/G and E129/A gp39 paint mutants showed that this residue does not contribute directly to CD40/gp39 binding bu t that its substitution with a glycine disrupts the gp39 structure. Co mparison of the gp39 and CD40 residues involved in receptor-ligand con tacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues f orm contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.