Abstract
The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.
Highlights
C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are members of the CSK family of protein tyrosine kinases
Intramolecular interactions between the Src Homology 2 (SH2) domain and tyrosine kinase domain are critical in regulation of catalytic activity
Studies have suggested that the SH3–SH2 linker interacts with the SH2 domain (Shekhtman et al 2001; Wong et al 2005; Mikkola and Gahmberg 2010), and this phenomenon was confirmed by our study
Summary
C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are members of the CSK family of protein tyrosine kinases. These proteins suppress the activity of Src family kinases (SFKs) by selectively phosphorylating the conserved C-terminal tail regulatory tyrosine (Nada et al 1991, 1993; Chong et al 2005, 2006). A disulfide bond in the SH2 is suggested to regulate Csk kinase activity (Mills et al 2007), the extent is possibly highly assay-specific The subcellular localization and activity of Csk are regulated by its SH2 domain (Chong et al 2005)
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