Abstract
The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.
Highlights
The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation
Combined crystallography and computational modeling have generated a molecular model for Src kinase activation, involving initial conversion of the autoinhibited kinase to a Tyr-416 unphosphorylated active-like state and subsequent phosphorylation of Tyr-416 resulting in stabilization of the active kinase[8,11,47,48]
Our conventional and metadynamics simulations of the entire autoinhibited Src protein show that Cys277-SOH destabilizes the folded activation loop (A-loop) containing Y416 toward a more favorable unfolded state on a shorter (~1.5–3.5 μs) timescale, which would imply that oxidation of Cys-277 accelerates the conversion to the active-like state, which is potentially stabilized in the absence of Tyr-416 phosphorylation (Fig. 4)
Summary
The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated. Recent studies indicate that redox-dependent activation of tyrosine kinases such as Src or EGFR closely associates with initial formation of Cys-SOH, the proximal product of cysteine oxidation by H2O2, and that other oxidative modifications such as S-glutathionylation do not enhance kinase activity[21,25,33,34]. Molecular dynamics (MD) and metadynamics simulations indicate that modification of these cysteines to Cys-SOH induces local structural changes that directly impact the regulatory pTyr-527 and Tyr-416 residues, respectively
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