Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation

Abstract

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser(132) loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.

FIGURE 1.

Overall structure of the CD40-CD154 complex. A, domain arrangements of human CD40 and CD154. The crystallized fragments are marked by red boxes and labeled. Top view (B) and side view (C) of the CD40-CD154 complex. The strands of CD154 are labeled. CRD1 and CRD3 of CD40 are colored dark blue, and CRD2 is colored light blue. D, molar ratio of CD40 and CD154 in solution. The purified complex of CD40 and CD154 were separated by SDS-PAGE after partial deglycosylation (see text). The protein bands were stained by Coomassie Brilliant Blue (left), excised, and quantitated by amino acid analysis (right). Samples from two independent preparations labeled as experiments 1 and 2 were analyzed. The minor band in the higher molecular weight region of the SDS-PAGE gel is that of peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase added for deglycosylation. It was not subjected to the quantitation analysis. Significant figures of the analysis represent accuracy of the final quantitation step of HPLC chromatograms.

FIGURE 2.

Structure of the CD40-CD154 interaction interface. A, the CD40 molecule is split from the bound CD154 and rotated to show the interaction interface. Residues directly contacting ligand or receptor are drawn. B, the CD40 contact area in CD154 and TNFR1 contact area in TNFβ are marked with broken lines and colored in gray. The contact areas can be divided into A and B patches (see text). C, residues making direct contacts are linked by broken lines, and residues found in hyper-IgM patients are boxed. The underlined residues were studied in previous mutagenesis research and proven to be critical for ligand-receptor interaction. D, surface representation of CD154 and CD40. The orientation is as in A. Positively and negatively charged surfaces are blue and red, respectively (left). Residues making critical charge interactions are linked by broken lines (right).

FIGURE 3.

Structural comparison of CD154 and CD40 with TNFβ and TNFR1. A, the structures of TNFβ and CD154 are aligned, and their Cα traces are superimposed. B, the structures of TNFR1 and CD40 are aligned. The side chains of Cys¹¹¹ and Cys¹¹⁶ forming a disulfide bridge are shown as ball-and-stick models. Sulfurs and carbons are in orange and black, respectively.

FIGURE 4.

CD40 binding activities of the CD154 mutants. A, positions of mutated residues in the crystal structure. The direct CD40 contact area in CD154 is marked with dashed lines. B, CD154 mutants used in the analysis. Equal amounts of affinity-purified wild type and mutant forms of soluble CD154 (residues Gly¹¹⁶–Leu²⁶¹) were analyzed by SDS-PAGE and detected by Coomassie Blue staining and immunoblotting with anti-CD154 antibody. C, flow cytometric analysis. HEK293T cells stably transfected with a vector carrying full-length CD40 receptor were treated with FACS buffer alone (Vehicle, see “Experimental Procedures”) or FACS buffer containing 10 μg/ml WT or mutant forms of CD154 protein for 30 min at 4 °C. An anti-CD40 antibody was used to verify CD40 expression in the stably transfected HEK293T cells. Binding with FITC-conjugated anti-FLAG antibody was analyzed by flow cytometry. The results are representative of more than three independent experiments.

FIGURE 5.

Reduced NF-κB activation and IL-6 production in the S132W mutant of CD154. The S132W mutant has substantially reduced activity in NF-κB activation and IL-6 secretion assays. A, HEK293T cells stably transfected with CD40 were transiently transfected with NF-κB reporter and incubated with or without CD154 WT or mutant proteins (5∼20 μg/ml) for 24 h. The cells were then subjected to the luciferase assay. B, human primary B cells were prepared as described in “Experimental Procedures,” incubated with or without CD154 wild type or mutant proteins for 7 days. IL-6 protein secreted into supernatants was measured by ELISA. C, THP-1 cells were treated with 10 μg/ml wild type or the indicated mutant forms of CD154 for 12 h and then subjected to semiquantitative RT-PCR analysis (left). Relative intensities of the ethidium bromide stained bands were analyzed by TINA2.0 (Strauben, Hardt, Germany) (right). Data are the means ± S.E. of three independent experiments.

FIGURE 6.

Reduced activation of p38 and ERK1/2 by the S132W mutant. The S132W mutant cannot activate p38 and ERK1/2. BJAB cells were stimulated with CD154 WT or mutant forms for 15 min at 37 °C. Cell lysates were analyzed by immunoblotting with antibodies against phosphorylated p38, ERK1/2, and JNK. The blots were stripped and immunoblotted again with antibodies to total p38, ERK1/2, and JNK. Relative intensities of the phosphor forms in the autoradiograms were analyzed by TINA2.0. Data are representative of three independent experiments.

FIGURE 7.

Effects of alanine insertions in the Ser¹³² loop of CD154. The loop-insertion mutant, S132–6A-K133, displays low NF-κB stimulating activity similar to the S132W mutant. A, HEK293T cells stably transfected with CD40 were incubated with CD154 wild type or mutant forms and analyzed by flow cytometry as in Fig. 4C. B, HEK293T cells stably transfected with CD40 were transiently transfected with an NF-κB reporter and incubated with or without CD154 wild type or mutant proteins for 24 h. Cells were then subjected to the luciferase assay. C, human primary B cells were prepared as described under “Experimental Procedures” and incubated with or without CD154 wild type or mutant proteins for 7 days. IL-6 secretion was measured by ELISA. D, BJAB cells were incubated with CD154 wild type or mutant proteins for 15 min at 37 °C. Cell lysates were analyzed by immunoblotting with antibodies to phosphorylated or total p38 as in Fig. 6. Data are the means ± S.E. of three independent experiments.