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 and CD40 on antigen-presenting cells play an important role in regulating antibody production by B cells, cytokine production by monocytes, and other immune responses which require T cell ''help''. Using structure-based sequence alignments, a molecular model of gp39, site-directed mutagenesis, and receptor-ligand binding assays, we have identified CD40 and gp39 surface residues which are important for receptor-ligand binding. Binding studies with CD40 or gp39 proteins containing single and double amino acid substitutions showed that CD40 residues Y82, D84, and N86 are involved in gp39 binding, while gp39 residues K143 and Y145 are important for CD40 binding. Analysis of the location of amino acid 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 binding. Binding studies with E129/G and E129/A gp39 paint mutants showed that this residue does not contribute directly to CD40/gp39 binding but that its substitution with a glycine disrupts the gp39 structure. Comparison of the gp39 and CD40 residues involved in receptor-ligand contacts with those previously identified as playing an important role in TNF-beta/TNFR binding suggests that some of the identified residues form contacts similar to those found in the TNF-beta/TNFR while others are unique to the CD40-gp39 interaction.