last update 2000/4/30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

On Going Research

[Bruton's Tyrosine Kinase] [Hepatitis delta virus] [DNA/RNA Hybrid]
[Adapter protein-Grb2] [Tetratricopeptide repeat-TPR]
  • Bruton's Tyrosine Kinase

    • The cytoplasmic tyrosine kinase (Bruton's tyrosine kinase, BTK) was found to play a central role for B cell development. Mutations or deletions within this protein are responsible for X-linked agammagobulinemia (XLA), an inherited immunodeficiency decease. BTK contains an apparent pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, a SH3 domain and a catalytic tyrosine kinase domain. SH2 and SH3 domains are small protein modules that mediate protein-protein interactoins and are found in many proteins involved in intracellular signal transduction. In order to investigate the role of BTK in B cell development and activation, we are applying multi-dimensional NMR techniques to study structures of the SH2 and SH3 domains of BTK. Peptide libraries will be collected to reveal novel binding ligands for the SH2 and SH3 domains of BTK. Structural studies of the ligand/SH2 and ligand/SH3 complexes will be carried out and new ligands can be designed through a rational approach.

    Related publications:
    1. Stability and Folding of the SH3 Domain of Bruton's Tyrosine Kinase (1996) PROTEINS: Structure, Function, and Genetics 26, 465-471.

    2. SH3 Domain of Bruton's Tyrosine Kinase can Bind to Proline-Rich Peptides of TH Domain of the Kinase and p120cbl (1997) PROTEINS: Structure, Function, and Genetics 29, 545-552.

    3. Solution Structure of the BTK SH3 Domain Complexed with a Proline-Rich Peptide from p120cbl (2000) Journal of Biomolecular NMR 16, 303-312.

    4. Stability and Peptide Binding Specificity of BTK SH2 Domain: Molecular Basis for X-Linked Agammaglobulinemia (2000) Submitted.

    5. Solution Structure and Backbone Dynamics of a Self-Associated TH-SH3 Domain from Bruton's Tyrosine Kinase (2000) in preparation.

    6. Events of the Folding Dynamics of the BTK SH3 Domain (2000) in preparation.

    7. Solution Structure of the BTK SH2 Domain: Structural Basis for X-Linked Agammaglobulinemia (2000) in preparation.

  • Adapter protein-Grb2

    • Growth factors, when binding to the external domain of their receptors, induce oligomerization of receptors, stimulation their protein kinase activity that is responsible for reciprocal transphosphorylation of receptor intracellular domains. The tyrosine phosphorylation sites exhibit a high affinity for SH2 domains (Src Homology 2 domain, ~100 amino acids), the specificity being determined by the residues immediately surrounding the phosphorylated tyrosine. The SH2 domain of Grb2 binds phosphotyrosyl peptides with the consensus sequence pYXNX within several proteins including the adapter proteins SHC, growth factor receptors such as members of the erbB family, morphology-determining proteins such as FAK, and cellular oncogenes such as BCR-abl. Binding of the Grb2 SH2 domain to the receptors relocates the Grb2 SH3 domain binding proteins, i.e. Sos, close to the plasma membrane. Sos, then, due to its guanine nucleotide exchange activity, converts the GDP-bound inactive form of Ras to its GTP-bound active form. Activated Ras triggers the kinase cascade which is essential for cell growth and differentiation. A particularly important role for Grb2 in human cancer has been proposed for cells transformed by high levels of erbB2 (HER-2 or neu) expression. Recent studies have indicated that Grb2 function is required for cell transformation by the neu and bcr-abl oncogenes. Thus, the design of specific inhibitors to Grb2 SH2 domain holds the promise of targeted treatment of breast cancer and cancer. We will use the structure-based drug design (SBDD) strategies to design and synthesize various inhibitors for the Grb2 SH2 domain.

    Related publications:
    1. Y.C. Lou, F.T. Lung, M.T. Pai, S.R. Tzeng, P.P. Roller and J.W. Cheng, "Solution Structure and Dynamics of a Nonphosphorelated Cyclic Peptide Inhibitor for the Grb2 SH2 Domain" (1999) Archives of Biochemistry and Biophysics 372, 309-314. [PDF format available]

     

  • Tetratricopeptide repeat-TPR

    • We will determine the solution structure and dynamics of the Tetratricopeptide repeat motif (TPR) of the Hsc70 associated protein - SGT using NMR spectroscopy. Binding surface and complex structure of the SGT TPR motif and Hsc70 will also be mapped using isotope-edited and filtered NMR experiments.

    Related publications:
    1. M.T. Pai, C.S. Yang, S.R. Tzeng, C. Wang, and J.W. Cheng, "Stability and Folding of the Tetratricopeptide repeat motif of SGT" (2000) in preparation.

     

  • Hepatitis Delta Antigen

    • Hepatitis delta virus (HDV) is a satellite of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. The genome of the hepatitis delta virus consists of a single-stranded, circular RNA of 1679 nucleotides which forms a rod structure due to extensive self homology. HDV replicates through synthesis of an antigenomic RNA via a rolling circle mechanism. This mechanism is governed by autocatalytic cleavage and ligation reactions. HDV encodes two proteins, the small delta antigen and the large delta antigen. The latter resembles the former except for the presence in the latter of additional 19 amino acids at the C terminus. While the small delta antigen is required for HDV RNA replication, the large delta antigen inhibits replication. HDV delta antigen differs from other RNA-binding proteins in that this antigen contains multiple regions (residues 2-27, 24-75, 79-107) that mediate RNA binding. In order to study the interaction of hepatitis delta antigen with HDV RNA, we will study the structure of its RNA-binding domain using CD and NMR spectroscopic techniques.

    Related publications:
    1. Y.C. Lou, I.J. Lin, M.T. Pai, and J.W. Cheng, "Solution Structure of an N-Capping Peptide from the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen" (2000) Archives of Biochemistry and Biophysics in press.

    2. I.J. Lin, Y.C. Lou, M.T. Pai, H.N. Wu and J.W. Cheng, "Solution Structure and RNA Binding Activity of the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen" (1999) PROTEINS: Structure, Function, and Genetics 37, 121-129.

    3. J.W. Cheng, I.J. Lin, Y.C. Lou, M.T. Pai, and H.N. Wu, "Local Helix Content and Nucleic Acid Binding Activity of the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen" (1998) Journal of Biomolecular NMR 12, 183-188.

     

  • DNA/RNA Hybrid

    • The solution structure of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]2, in which the central segment was flanked by DNA duplexes at both ends, was determined using 2D NMR, restrained MD, and NOE back-calculation refinement. Evidence of hydration at different sites in both grooves was found in NOESY and ROESY experiments. Correlation times of hydration and dissociation rate constants between the OH protons and water were measured. The solution structure of this chimeric duplex differed from previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]2 as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]2. Overall, the global conformation of this chimeric duplex was closer to its A-RNA analog than to its B-DNA analog . Furthermore, NOEs between water and H1ĦĤin the minor groove, which was not observed in its DNA analogue, showed similar identities with its RNA analogue. In the chimeric fragment, no structural parameter of the 5'-end DNA at the DNAĦERNA hybrid junction was affected by the 3'-end RNA, whereas structural change was found at the 3'-end RNAĦEDNA hybrid junction. This influence was involved in only one step. In contrast to the similarity with its RNA analog, titration of the minor groove binding drug, distamycin A suggested a possible 2:1 binding mode similar with previous DNA-drug binding studies. This result suggested a wider minor width, enough for two parallel-binding mode found in pure DNA duplex. Further structural studies are underway.

    Related publications:
    1. S.T. Hsu, M.T. Chou, and J.W. Cheng, "The Solution Structure of [d(CGC)r(aaa)d(TTTGCG)]2: Hybrid Junctions Flanked by DNA Duplexes" (2000) Nucleic Acids Research 28, 1322-1331.

    2. S.T. Hsu, M.T. Chou, S.H. Chou, W.C. Huang, and J.W. Cheng, "Hydration of [d(CGC)r(aaa)d(TTTGCG)]2" (2000) Journal of Molecular Biology 295, 1129-1137.

    3. Y.P. Tsao, S.T. Hsu, S.H. Chou, and J.W. Cheng, "Solution Structure and Hydration of [d(CGC)r(amamam)d(TTTGCG)]2" (2000) in preparation.