The role of polar interactions in the molecular recognition of CD40L with its receptor CD40

Abstract

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 isotype switching of the B-cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein 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 have used a combination of molecular modeling, mutagenesis, and X-ray crystallography. The structure of the extracellular region of CD4OL was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X-ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD4OL presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, El 14, E l 17). These residues were studied experimentally through site-directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, El14A, E l 17A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, E l 14R, El 17R of CD40), mutation to a polar residue (K143N, R207N, R207Q of C340L, and D84N, E l 17N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge-reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74-R203, D84-R207, and El 17-R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNFjTNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.

Different molecular behavior of CD40 mutants causing hyper-IgM syndrome

“…Based on structural data, the region affected by the mutation is not directly involved in binding to CD40L. 36 Consistently, we have demonstrated that P5 BLCL and P5 HEK-293 transfectants can bind recombinant CD40L. Moreover, upon stimulation with CD40L, HEK-293 cells expressing the P5 mutant can activate 2 independent NF-B signaling pathways.…”

A TNF receptor loop peptide mimic blocks RANK ligand–induced signaling, bone resorption, and bone loss

“…The presence in RANK of key features of the TNFR(I) CRD3 contact point, particularly Trp107 and Ser108 ( Figure 1A), suggested that the small cyclic WP9QY peptide modeled on the TNFR(I) CRD3 loop, which binds TNF-α and inhibits TNF-α-induced cytotoxicity (26), might also bind RANKL and alter RANKL-induced cell responses. The highly conserved folding of members of the TNFR and TNF superfamilies allows the structural modeling of receptor-ligand complexes that have not yet been experimentally characterized (28,29). We therefore used computer modeling to initially examine the possibility that the WP9QY peptide could bind to the RANKL trimer and thereby affect the RANKL-RANK complex.…”

Immune regulation by CD40-CD40-L interactions - 2; Y2K update

“…More recently the modularity of the TNF-receptor family has been refined, suggesting that every domain is built of two modules, with 5 different forms of modules being identified (13). Although the structure of CD40 has not been resolved, a model has been built using homology modeling, mutagenesis, X-ray structures of TNF-R and allignment of the TNF-R family (14). The mouse CD40 gene, composed of nine exons that span a 16.3 kb of genomic DNA, is located on the distal region of chromosome 2 which is syntenic to human chromosome 20q11-q13 where the human CD40 gene is located.…”