Ming-Tao Pai

Local helix content and RNA-binding activity of the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. Our results show that the N-terminal leucine-repeat region of hepatitis delta antigen (HDAg), encompassing residues 24–50, binds to the autolytic domain of HDV genomic RNA and attenuates its autolytic activity. The solution conformation of a synthetic peptide corresponding to residues 24–50 of HDAg as determined by two-dimensional 1H NMR and circular dichroism techniques is found to be an α-helix. The local helix content of this peptide was analyzed by NOEs and coupling constants. Mutagenesis studies indicate that Lys38, Lys39, and Lys40 within this α-helical peptide may be directly involved in RNA binding. A structural knowledge of the N-terminal leucine-repeat region of HDAg thus provides a molecular basis for understanding its role in the interaction with RNA.

Solution structure and RNA-binding activity of the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. The RNA genome of HDV encodes two proteins: the small delta antigen and the large delta antigen. The two proteins resemble each other except for the presence of an additional 19 amino acids at the C terminus of the latter species. We have found that the N‐terminal leucine‐repeat region of hepatitis delta antigen (HDAg) binds to the autolytic domain of HDV genomic RNA and attenuates its autolytic activity. A 27‐residue polypeptide corresponding to residues 24–50 of HDAg, designated dAg24–50, was synthesized, and its solution structure was found to be an α‐helix by circular dichroism and 1H‐nuclear magnetic resonance (NMR) techniques. Binding affinity of dAg24–50 with HDV genomic RNA was found to increase with its α‐helical content, and it was further confirmed by modifying its N‐ and C‐terminal groups. Furthermore, the absence of RNA binding activity in the mutant peptides, dAgM24–50am and dAgMAc24–50am, in which Lys38, Lys39, and Lys40 were changed to Glu, indicates a possible involvement of these residues in their binding activity. Structural knowledge of the N‐terminal leucine‐repeat region of HDAg thus provides a molecular basis for the understanding of its role in the interaction with RNA. Proteins 1999;37:121–129.

NMR based solution structure and dynamics of a nonphosphorylated cyclic peptide inhibitor for the Grb2 SH2 domain

Grb2 is an adaptor protein with a domain structure of SH3-SH2-SH3. Grb2 SH2 binds pTyr-peptides with the consensus sequence pYXNX within several proteins. Binding of Grb2 SH2 to receptors triggers the kinase cascade which is essential for cell growth and differentiation. The design of Grb2 SH2 inhibitors holds the promise of targeted inhibition of this pathway. Recently, we discovered a nonphosphorylated cyclic peptide ligand, G1TE, for the Grb2 SH2 domain [1,2]. Nonphosphorylated G1TE defines a new type of SH2 domain binding motif that may advance the design of Grb2 inhibitors. In order to gain further insight into these specific protein-protein interactions, we have determined the solution structure and dynamics of G1TE using two dimensional NMR and isotope labeling techniques. Results of conformational studies provide a molecular basis for the structurebased design of Grb2 SH2 inhibitors.

Structure and Peptide Binding Specificity of the SH3 and SH2 Domain from a Self-Regulated Protein Tyrosine Kinase — BTK

The cytoplasmic tyrosine kinase, Bruto’s tyrosine kinase (BTK), was found to play a central role in B cell proliferation and differentiation. BTK, along with Tec, Ltk and Atk, belong to a small family of tyrosine kinases (the Tec family) that share common structural features. However, little is known about their biological roles. BTK contains an N-terminal PH domain, an SH2 domain, an SH3 domain and a kinase domain. Mutations or deletions within these domains are responsible for X-linked agammaglobulinemia (XLA), an inherited immunodeficiency disease [1]. The SH3 and SH2 domains are small protein modules that mediate protein-protein interactions and are found in many proteins involved in intracellular signal transduction. It was shown that the BTK SH2 domain is essential for phospholipase C-γ phosphorylation; 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-γ [2,3]. However, the molecular basis for the interaction between the BTK SH2 domain and BLNK and the cause of XLA remain unclear. Although the structures of BTK SH3 domain and its complex with proline-rich peptides have been solved by NMR [4,5], little is known about the structure and peptide binding specificity for the SH2 domain of BTK. In order to understand the role of BTK in B cell development, in here, we report the structure and peptide binding specificity of the SH3 and SH2 domains of the self-regulated protein tyrosine kinase — BTK.

Folding and Peptide Binding of TPR Motifs from SGT

In eukaryotes, many proteins have evolved repetitive motifs to perform functions specific to their unique physiological demand. Mostly, these motifs have length between 20 and 40 residues and mediate a variety of distinctive protein-protein interactions [1]. These motifs often exist as a tandem array between 3 to 25 units to form a characteristic structure. Secondary structure of many of these motifs are found to be α-helices. For example, the armadillo repeat of 42 residues forms three α-helices, the ankyrin repeat of 33 residues adopts a β-hairpin-helix-loop-helix (β2α2) fold, the HEAT motif of 37–43 residues consists two antiparallel helices, and the tetratricopeptide repeat (TPR) motif of 34 residues forms an antiparallel couple of helices. Generally, these motifs have little conservation between their sequences, however, they all adopt compact conformations in all structures studied until now.