A Novel Disulfide Bond in the SH2 Domain of the C-terminal Src Kinase Controls Catalytic Activity

Abstract

SummaryThe SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond not present in other known SH2 domains. To investigate whether this unusual disulfide bond could serve a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small-lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteines in the SH2 domain greatly impacts catalytic function and domaindomain communication in Csk. KeywordsCsk; cysteine; disulfide; kinase; NMR; phosphorylation The Src family of tyrosine kinases [SFKs] 1 are modular enzymes responsible for controlling various aspects of cellular growth and differentiation.[1] SH3 and SH2 domains in the SFKs flank the kinase domain and repress catalytic activity through intrasteric constraints. Most notably, the SH2 domain interacts tightly with a phosphotyrosine in the C-terminus of the kinase domain, inducing a closed conformation that poorly phosphorylates protein substrates.

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

“…As shown earlier, Csk is marginally sensitive to redox modulation because of the formation of an intramolecular disulfide bond in the SH2 domain (25). Csk contains a Glu residue, Glu-205, at the position equivalent to Cys-277 of Src.…”

“…First, ROS may regulate any of the upstream regulatory kinases, phosphatases and G proteins, which would indirectly regulate Src activity. For example, the SH2 domain of Csk contains a novel disulfide bond, which modulates the Csk activity (25). By regulating Csk activity, ROS could indirectly alter Src activity.…”

“…1 A). The modest activation of Csk by DTT is caused by the reduction of a disulfide bond between Cys-122 and Cys-164 located in the SH2 domain (25). We also tested whether Src activity responds to the presence of H 2 O 2 .…”

Allostery in the ferredoxin protein motif does not involve a conformational switch

“…Transdomain "cross-talk" between a catalytic center and a distal loop is an emerging theme by which single domains can transmit cellular signals (7,(53)(54)(55). In the single-domain protein interleukin-1β, point mutations that are distal to the receptor binding interface have no effect on stability, yet they have significant effects on the binding properties (55).…”

“…In multidomain proteins, relative repositioning of domains is often linked to their levels of activity. For example, large-scale domain rearrangements in the four-domain Src kinase (4) and C-terminal Src kinase (5,6) lead to these proteins being in so-called "on" or "off" states, and functional regulation may be obtained by adjusting the balance between these conformations (7). In proteins such as adenylate kinase, domain rearrangements can be rate limiting during each round of catalysis (8), where the enzyme cycles between ligandcompetent and ligand-release conformations.…”