title: Dynamics of the Tec-family tyrosine kinase SH3 domains.
abstract: The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, 02115.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Roberts
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15219.
authors.forename: Sreya
authors.initials: S
authors.lastname: Tarafdar
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, 50011.
authors.forename: Raji E
authors.initials: RE
authors.lastname: Joseph
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, 50011.
authors.forename: Amy H
authors.initials: AH
authors.lastname: Andreotti
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15219.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, 02115.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, 02115.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wales
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D030561:Databases, Nucleic Acid; D041961:Deuterium Exchange Measurement; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics*; D018909:src Homology Domains
chemical_list: D011505:Protein-Tyrosine Kinases
keywords: Bruton's tyrosine kinase (Btk); EX1 kinetics; IL-2 inducible T cell kinase (Itk); hydrogen exchange mass spectrometry