ANALYSIS OF GP39 CD40 INTERACTIONS USING MOLECULAR-MODELS AND SITE-DIRECTED MUTAGENESIS/

The interaction between gp39 (CD40L, TRAP, T-BAM) on activated T cells and mast cells and CD40 on antigen-presenting cells modulates immune responses. Gp39 and CD40 are homologous to tumor necrosis factor (TNF) and its receptor (TNFR), respectively. The TNF-beta/TNFR interaction has been analyzed on the basis of mutagenesis experiments and crystal structures. Using the interaction of TNF-beta/TNFR as a guide, we prev iously reported a site-directed mutagenesis study in which we identifi ed residues in gp39 (K143, Y145) and CD40 (Y82, D84, N86) involved in gp39/CD40 interactions. Here we describe the use of the TNF-beta/TNFR complex crystal structure as a template to prepare molecular models of gp39, CD40, and their approximate interaction. The application of the se models has allowed us to extend our mutagenesis analysis of gp39/CD 40 interactions, These experiments have led to the identification of a dditional gp39 (Y146, R203, Q220) and CD40 (E74, E117) residues that c ontribute to the gp39/CD40 interaction. We also further explored the i mportance of gp39 residue Y145 and CD40 residue Y82 for the gp39/CD40 interaction by conservatively replacing these residues with Phe. The r esults of these studies have enabled us to approximately outline the b inding sites in gp39 and CD40. It appears that the gp39/CD40 interacti on is centered on at least two clusters of residues and involves resid ues of two adjacent gp39 monomers. The molecular regions involved in t he gp39/CD40 interaction essentially correspond to those in the homolo gous TNF-beta/TNFR system.