Crystallographic and Mutational Analysis of the CD40-CD154 Complex and Its Implications for Receptor Activation

Hyun-Jung An,Young Jin Kim,Dong Hyun Song,Beom Suk Park,Ho Min Kim,Ju Dong Lee,Sang-Gi Paik,Jie-Oh Lee,Hayyoung Lee

doi:10.1074/jbc.m110.208215

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.

Highlights

Expressed on activated T lymphocytes and regulates B cell function by binding to CD40 on the B cell surface
Because CD154, like other TNF family proteins, exists as a stable trimer [11], it has been proposed that its binding leads to trimerization of CD40 and initiates intracellular signaling by recruiting TRAF4 adapter proteins to the cytoplasmic domain of CD40 [9]
Mutagenesis studies have demonstrated that the intracellular region of CD40 has multiple TRAF binding sites

Summary

Introduction

Expressed on activated T lymphocytes and regulates B cell function by binding to CD40 on the B cell surface. CD154 is a ϳ30-kDa type II transmembrane protein containing a TNF homology domain responsible for receptor binding and activation [7] It has been detected in supernatants of activated T cells as soluble forms with apparent molecular masses of 14, 18, and 29 kDa [13, 14]. Crystal Structure of CD40-CD154 Complex (TNF-related apoptosis-inducing ligand), and BAFF (B cell-activating factor), complexed with their cognate receptors have been reported [15,16,17,18,19]. All these TNF receptor (TNFR) family proteins contains several cysteine-rich domains (CRDs) forming an elongated ladder-like structure. Mutagenesis experiments of CD154 led us to identify a loop that is critical for receptor activation but not for receptor binding

Methods

Results

Conclusion