A Highly Conserved Cysteine of Neuronal Calcium-sensing Proteins Controls Cooperative Binding of Ca2+ to Recoverin

Abstract

Background: Recoverin contains a cysteine (Cys-39) that is highly conserved in neuronal calcium-sensing (NCS) proteins. Results: The C39A mutation shifts the conformational equilibrium from the R to T state, inducing cooperative calcium binding. Conclusion: Cys-39 controls the conformational equilibrium of calcium-free recoverin. Significance: This mutation assigns a previously unknown function to the conserved cysteine in recoverin and possibly all NCS proteins.

Biological chemistry and functionality of protein sulfenic acids and related thiol modifications

“…Mild oxidative environments regulate the activity of recoverin by acting on a conserved Cys residue (Cys39 in the human homolog) [195–197]. Experiments using site-directed mutagenesis of Cys39 to Asp (-SO 2 H mimetic) showed that hyperoxidation of myristoylated protein did not change the affinity for Ca 2+ but drastically decreased the binding of recoverin to photoreceptor cell membranes [196].…”

“…Experiments using site-directed mutagenesis of Cys39 to Asp (-SO 2 H mimetic) showed that hyperoxidation of myristoylated protein did not change the affinity for Ca 2+ but drastically decreased the binding of recoverin to photoreceptor cell membranes [196]. On the other hand, the Cys39Ala mutant increased the cooperativity of Ca 2+ binding to non-myristoylated protein [197]. Structural analysis further identified –SOH modification at Cys39 [197].…”

“…On the other hand, the Cys39Ala mutant increased the cooperativity of Ca 2+ binding to non-myristoylated protein [197]. Structural analysis further identified –SOH modification at Cys39 [197]. Thus, the oxidative modification of this protein controls its Ca 2+ binding properties, intracellular location and potentially the interaction with other proteins involved in the visual cycle of photoreceptor cells.…”

Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands

“…All proteins were expressed in and purified from T7 Express Escherichia coli (New England Biolabs) as described previously 14 . WT and mutant Rv proteins were prepared in the non-myristoylated form.…”

“…Ca 2+ -binding assays were performed in triplicate according to previously established methods 11, 14 using a Hitachi F-2500 fluorescence spectrometer (Tokyo, Japan) to monitor Ca 2+ -induced changes in intrinsic tryptophan fluorescence. Titration data were fit to a model for two independent sites $$f\left({normal\mathit{Ca}}^{2+}\right)=0.5true(\frac{\left[{normal\mathit{Ca}}^{2+}\right]}{\left[{normal\mathit{Ca}}^{2+}\right]+K_1}+\frac{\left[{normal\mathit{Ca}}^{2+}\right]}{\left[{normal\mathit{Ca}}^{2+}\right]+K_2}true)$$ where [ Ca 2+ ] is the free Ca 2+ concentration, f(Ca 2+ ) is the fraction of total sites occupied by Ca 2+ , and K 1 and K 2 are the first and second dissociation constants, respectively.…”