Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


osu1324406526.pdf (3.63 MB)

ETD Abstract Container

Abstract Header

Sequence Specificity of Src Homology-2 Domains

Tan, Pauline H.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1324406526

Abstract Details

2012, Doctor of Philosophy, Ohio State University, Chemistry.
Src-homology domains are small modular domains that recognize phosphotyrosine-containing proteins and couple activated protein kinases to intracellular signaling pathways. Since they often have overlapping functions, their SH2 domains often compete for binding to the same pY proteins. Determining their sequence specificities will help identify target proteins. Consequently, this will help understand the molecular basis for their cellular functions. Twenty-six kinase SH2 domains were screened against a phosphotyrosyl (pY) peptide library, and positive beads were sequenced by partial Edman degradation and mass spectrometry. The data revealed that the kinase family SH2 domains selected a class of pY peptides consisting of mostly hydrophilic and hydrophobic residues at the pY+1 and pY+3 positions, respectively. After validating their binding, the literature was searched to find known SH2 targets and their pY motifs. Seventeen SH2 domains from several different protein families were also purified, screened, and sequenced to determine their binding motifs. The majority of the SH2 domains had high selectivity at the pY+3 or pY+1 position with a few selecting for multiple peptide classes. For example Vav1 and Vav2 SH2 domains selected for three classes of peptides. These minor classes of peptides may be motifs of new protein targets that have not been identified yet. Some SH2 domains such as the Grb7 family, HSH2D, and Vav family had high selectivity of Asn at the +2 position but little selectivity at other positions. More subtle differences were observed between protein families at certain positions. For instance, SH2 domains from a family of GTPase signaling proteins preferred Pro at the +3 position, while SH2 domains from the PIK3 family preferred norleucine at the +3 position. Genetic disorders such as Noonan’s syndrome and hematologic disorders such as juvenile myelomonocytic leukemia are caused by mutations in the SHP2 (PTPN11) gene. The mutations are mainly located in the SH2 binding cleft or in the NSH2/PTP interface. Mutations in the SH2 binding cleft may alter sequence specificity, resulting in new potential binding partners. Identifying novel binding partners may help lead to a better understanding of the complex mechanism of these disorders. Five SHP2 SH2 mutants were screened against a combinatorial phosphotyrosyl (pY) peptide library, and positive hits were sequenced. Only the T52S SH2 mutant exhibited a specificity switch from small to large branched hydrophobic residues at the +1 position. The T42A and L43F SH2 mutants exhibited increased binding affinities. In contrast, the specificities and binding of E76K and E139D SH2 mutants remained unchanged. After examining the crystal structure generated by PyMOL, altered specificity involved a substitution of a bulky Thr with a smaller Ser residue allowing more space for larger branched residues. The T52S consensus was entered into a protein database to search for new protein targets that bind to the mutant only. Twenty-six potential targets resulting from the database search were identified for the T52S mutant.
Dehua Pei, PhD (Advisor)
Jennifer J. Ottesen, PhD (Committee Member)
Karin Musier-Forsyth, PhD (Committee Member)
167 p.
Biochemistry; Chemistry
SH2 Domain; Binding Specificity; Src kinase; kinase; SHP2; SH2 mutant; protein-protein interaction; peptide library; protein binding;

Recommended Citations

Citations

  • Tan, P. H. (2012). Sequence Specificity of Src Homology-2 Domains [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1324406526

    APA Style (7th edition)

  • Tan, Pauline. Sequence Specificity of Src Homology-2 Domains. 2012. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1324406526.

    MLA Style (8th edition)

  • Tan, Pauline. "Sequence Specificity of Src Homology-2 Domains." Doctoral dissertation, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1324406526

    Chicago Manual of Style (17th edition)

osu1324406526
789
© 2011, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.