Skip to Main Content
 

Global Search Box

 
 
 
 

ETD Abstract Container

Abstract Header

Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT Domains

Hard, Ryan Lawrence

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Biochemistry.
Src Homology 2 (SH2), Post-Synaptic Density-95/Discs Large/Zonula Occludens-1 (PDZ), Binder of Ubiquitin Zinc Finger (BUZ), and BRCA1 C-terminal (BRCT) domains are short peptide-binding modules that recognize different types of peptide motifs within their protein binding partners. Determination of the type of peptide motifs preferred by each domain would lead to a better understanding of the in vivo role of each domain, including which protein(s) they recognize and how they recognize them. To determine the binding specificity of these domains, we constructed combinatorial one-bead-one-compound (OBOC) peptide libraries and screened them against each domain, sequenced positive hits from the screens by partial Edman degradation/mass spectrometry, and sorted the binding sequences into recognizable patterns. We used in vitro peptide binding assays, site-directed mutagenesis, and pull-down assay techniques to validate the screening results and help understand the nature of the protein-peptide interactions. The SH2 domains of PLCG1 (both SH2 domains) and TSAd were screened against phosphotyrosine (pY)-containing OBOC peptide libraries. The SH2 domain of TSAd selected for only one class of sequences, while the other SH2 domains each selected multiple classes of ligands. Generally, the SH2 domains showed selectivity for residues at the +1, +2, and +3 positions (relative to pY) and not for residues N-terminal to pY. PDZ and BUZ domains recognize their protein binding partners at the C-terminus, specifically requiring the free carboxylate group for efficient binding. In order to probe the binding specificity of these C-terminal binding domains, we constructed spatially segregated OBOC peptide libraries, where peptides in the exterior of each bead presented a free C-terminus and could therefore interact with the PDZ and BUZ domains, while peptides in the bead interior presented a free N-terminus so that they could be sequenced using partial Edman degradation and mass spectrometry. Using this type of library, the C-terminal sequence specificity of the PDZ domains of Tiam1 and Tiam2 and the BUZ domains of Ubp-M and HDAC6 were examined. Each PDZ and BUZ domain selected for multiple classes of binding sequences. In vitro peptide binding studies were used to verify the BUZ/PDZ-peptide interactions, including ones with a Tiam1 mutant that helped to illustrate the basis of PDZ domain ligand affinity and specificity. Furthermore, the BUZ domain of Ubp-M was used in a pull-down assay of a protein containing a C-terminus matching one of its consensus binding sequences, which further validated the screening results. Tandem BRCT domains recognize their protein binding partners either at internal or C-terminal peptide motifs. In either case, they usually require at least one phosphoserine (pS) or phosphothreonine (pT) residue to bind with high affinity. In order to determine the phosphopeptide binding specificity of the tandem BRCT domains, we constructed two pS/pT OBOC libraries. One pS/pT OBOC library, like the libraries used to study the PDZ and BUZ domains, presented peptides with free C-termini on the library bead surfaces and contained encoding peptides with free N-termini in the bead interior. A second library was constructed where the peptides were presented in the normal N-terminal to C-terminal direction on both the interior and exterior portions of the library beads. All 16 known human tandem BRCT domains (or their closely related mouse orthologs) were screened against both phosphopeptide libraries, with 8 of the 16 domains giving either well-defined or general consensus binding motifs. The C-terminal BRCT repeat of PTIP was found to bind to a novel dual phosphoserine motif. The remainder of the domains either failed to show binding selectivity or showed binding selectivity in the peptide library screens but failed to bind to resynthesized peptides in solution.
Michael Ibba, Dr. (Committee Chair)
Jennifer Ottesen, Dr. (Committee Chair)
Dehua Pei, Dr. (Advisor)
239 p.

Recommended Citations

Citations

  • Hard, R. L. (2013). Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT Domains [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1377005582

    APA Style (7th edition)

  • Hard, Ryan. Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT Domains. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1377005582.

    MLA Style (8th edition)

  • Hard, Ryan. "Sequence Specificity of BUZ, PDZ, SH2, and Tandem BRCT Domains." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1377005582

    Chicago Manual of Style (17th edition)