Abstract
Bruton's tyrosine kinase (Btk) is encoded by the gene that causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice when mutated. The SH2 domain, the fourth domain of the human Btk, is essential for phospholipase C-□ phosphorylation and mutations in this domain lead to XLA. Recently, the B-cell linker protein (BLNK, also called SLP-65) was found to interact with the SH2 domain of Btk and this association is required for the activation of phospholipase C-□. To understand the role of Btk in B cell development, here we have reported the 1H, 15N, and 13C resonance assignments of the BTK-SH2 domain and have determined the solution structures and the human BLNK phosphopeptide binding sites of the Btk SH2 domain. We have also found that mutation sites of the Btk SH2 domain identified from XLA patients are involved in the phosphopeptide binding. It is likely that the point-mutated Btk SH2 domains fail to present to the ligand the crucial residue in the correct context, thus leading to a weaker binding. The altered binding behavior likely affects the kinase function, and possibly causes XLA. In addition to the isolated Btk SH2 domain, we have studied the structure and mechanism of the self-regulation phenomenon of a protein fragment containing the TH-SH3-SH2 domain of Btk by NMR spectroscopy and gel permeation chromatography. Chemical shift perturbations indicate that residues located in the RT loop, n-Src loop and helix-like loop between □4 and □5 of the SH3 domain exhibit large changes. These chemical shift changes are in a manner similar to those have been found in the p120cbl proline-rich peptide/SH3 complex. The results indicate an intermolecular association through the proline-rich region of the TH domain and the PXXP binding sites of the SH3 domain but not through the SH3 and SH2 domain-domain interactions. Based on the present and previous studies, we propose a model for the control of the signaling processes of Btk in B cell development. Dengue is an acute infectious disease caused by the dengue virus (DENV), which has four serotypes. The virion contains three structural proteins - a 12 kD nucleocapsid or core protein (C), a 8 kD non-glycosylated membrane protein (M), and a 53 kD glycosylated envelope protein (E), as well as seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). DENV initiates infection by attaching to host cells via interaction between viral surface proteins and specific receptor/coreceptor molecules on target cells. The outer domain III of the dengue virus envelope protein (DENV-E3) is the dominant antigen in eliciting neutralizing antibodies and plays an important role in binding to target cell heparin sulfate. In order to provide the structural basis for immunologic protection and for vaccine design effective against DENV, we have determined the solution structure and the antibodies-neutralizing binding sites of the domain III of the dengue virus envelop protein.