THE ROLE OF POLAR INTERACTIONS IN THE MOLECULAR RECOGNITION OF CD40L WITH ITS RECEPTOR CD40

CD40 Ligand (CD40L) is transiently expressed on the surface of T-cells and binds to CD40, which is expressed on the surface of B-cells. This binding event leads to the differentiation, proliferation, and isotyp e switching of the B-cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L prote in causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T-cell dependent B-cell activation . To understand the structural basis for CD40L/CD40 association, we ha ve used a combination of molecular modeling, mutagenesis, and X-ray cr ystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was bui lt using homology modeling based upon a novel alignment of the TNF rec eptor superfamily, and using the X-ray structure of the TNF receptor a s a template. The model shows that the interface of the complex is com posed of charged residues, with CD40L presenting basic side chains (K1 43, R203, R207), and CD40 presenting acidic side chains (D84, E114, E1 17). These residues were studied experimentally through site-directed mutagenesis, and also theoretically using electrostatic calculations w ith the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R2 03A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge rev ersal (K143E, R203E, R207E of CD40L, and D84R, E114R, E117R of CD40), mutation to a polar residue (K143N, R207N, R207Q of C340L, and D84N, E 117N of CD40), and for the basic side chains in CD40L, isosteric subst itution to a hydrophobic side chain (R203M, R207M). All the charge-rev ersal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the c omplex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74-R203, D84-R207, and E117-R207 ha d a net stabilizing effect on the complex. However, the substitution o f hydrophilic side chains at several of the positions was tolerated, w hich suggests that although charged interactions stabilize the complex , charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identif ied a set of polar interactions surrounded by a wall of hydrophobic re sidues that appear to be similar but inverted between the two complexe s.