The B cell antigen receptor (BCR) is a multiprotein structure that consists of a non-covalently associated antigen binding subunit and a signaling subunit. Membrane-bound immunoglobulin (Ig) is an antigen binding subunit that has a very short cytoplasmic tail of only about three amino acids. Signal is transmitted through disulfide-linked heterodimer consisting of Igα and Igβ proteins (CD79a and CD79b). Igα and Igβ each contain a single immunoreceptor tyrosine-based activation motif (ITAM) within their cytoplasmic tail that initiates signal transduction following BCR aggregation. Aggregation of the BCR results in the phosphorylation of the ITAM tyrosine residues on Igα and Igβ primarily by src-family kinases. In efforts to elucidate molecular recognition between Iga and Igb and between the heterodimer and the BCR through structural studies, we have expressed both human and murine extracellular domains of Igα and Igβ in a recombinant E. coli system as inclusion bodies. Attempts were made to refold Igα and Igβ either individually or in concert. Both Igα and Igβ can be refolded individually. Refolding of Igα yielded a mixture of monomeric and homodimeric species as determined by mass spectrometry and SDS-PAGE analysis. The crystallization trials of Igα, however, was not successful. In contrast, the crystallization experiments of both the refolded human and murine Igβ yielded crystals that diffracted to 3.8 and 1.7 angstrom resolution, respectively. Subsequently, we have determined the structure of extracellular portion of murine Igβ at 1.7 ? resolution. The diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. The structure was solved by the molecular replacement method with the final R-factors are R=18.7 and Rfree=19.7%. As predicted, the structure shows a V-type immunoglobulin fold made of two antiparallel beta sheets with a characteristic A-strand switch and conserved disulfide bond between the B and F strands flanked by tryptophan from C strand. There are several distinct features in the murine Igβ structure. Among them, a new disulfide bond was observed connecting the N-terminus and the beginning of G strand. In addition, the typical C?, C? antiparallel strands characteristic for a V-type Ig fold are not present in murine Igβ. Instead of C?/C? strands, the structure shows a much shorter strand bridging two antiparallel beta sheets. There is one free cystein located in FG loop which is blocked by glutathione in the structure. Presumably, this cystein takes part in the formation of a disulfide bond in Igα/Igβ heterodimer. Further structural and binding studies are currently underway.? ? Complement pathway constitues an important part of host immune system against infection. The classical complement pathway is activated when immunoglobulins or the acute phase reactive protein CRP bind to C1q. Our goal is to understand the molecular mechanism of C1q activation. We are currently purifying C1q from human plasma, constructing recombinant expression system for the Fc regions of the immunoglobulins. We hope to be able to determine the crystal structure of C1q in complex with CRP or immunoglobulins. Recent evidence suggests that CRP interacts with Fc receptors raising the possiblity of cross interaction between the complement and the Fc receptor mediated pathways. We plan to investigate the interaction between CRP and Fc receptors and determine the structure of their complex.
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| Zhang, Y; Boesen, C C; Radaev, S et al. (2000) Crystal structure of the extracellular domain of a human Fc gamma RIII. Immunity 13:387-95 |
