The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

Abstract

Oncomodulin (Ocm), or parvalbumin β, is an 11-12 kDa Ca²⁺-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca²⁺-specific domains of the EF-hand type ("helix-loop-helix" motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pK_a of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol-disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca²⁺-loaded protein is of -168 mV and -176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg²⁺-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca²⁺/Mg²⁺ affinity of the EF site, and accelerates Ca²⁺ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca²⁺ affinity.

Keywords: EF-hand; calcium-binding protein; cysteine; disulfide dimerization; oncomodulin; parvalbumin; protein stability; redox potential; redox sensor; thiol oxidation.

Figure 1

pH dependence of molar extinction coefficient at 240 nm at 20 °C, after subtraction of contribution of the deprotonated Tyr residues, for apo- (open symbols) and Ca²⁺-loaded (solid symbols) forms of reduced rWT Ocm (circles) and C18S Ocm (triangles). Protein concentration was 23 μM. Buffer conditions: 30 mM MES, 30 mM HEPES, 30 mM H₃BO₃, 30 mM glycine, 100 mM KCl, 1.5 mM EDTA (apo-Ocm), or 1 mM CaCl₂ (Ca²⁺-bound Ocm).

Figure 2

Quantitation of redox equilibrium between apo-/Ca²⁺-loaded dOcm and GSSG/2GSH redox couple at 35 °C. 1.5 μM dOcm solution (50 mM HEPES-KOH, 100 mM KCl, 1 mM EDTA/CaCl₂, pH 7.4) was incubated under anaerobic conditions with 0.2 mM GSSG and 25 μM to 1.6 mM GSH for 62 h, followed by non-reducing SDS-PAGE. The weight fractions of dOcm were derived from quantitative analysis of the gels. The top axis corresponds to the redox potential of GSSG/2GSH redox pair (scheme [II]) calculated according to Equation (4). The dashed curves correspond to theoretical fits of the data computed according to scheme [I] using Equation (1).

Figure 3

Temperature dependencies of specific heat capacities of apo- and metal-bound states of rWT/C18S Ocm, derived from DSC data [46]. Protein concentration was 1.2–2.1 mg/mL. Buffer conditions: 10 mM H₃BO₃-KOH, 1 mM CaCl₂, pH 9.0 (Ca²⁺-bound Ocm), 20 mM glycine-KOH, pH 9.2, 1 mM EDTA (apo-Ocm), or 20 mM glycine-KOH, pH 9.2, 1 mM MgCl₂ (Mg²⁺-bound Ocm). Heating rate was 1 K/min. The DSC data are analyzed according to the cooperative two-state model [III]. The experimental and theoretical curves are shown solid and dashed, respectively. The dotted curve corresponds to specific heat capacity of the fully unfolded rWT Ocm, calculated according to ref. [48].

Figure 4

Fluorimetric Ca²⁺ and EDTA titration of rWT/C18S Ocm (a) and time course of EGTA-induced Ca²⁺ removal from Ocm monitored by fluorescence stopped-flow technique (b) at 20 °C. Buffer conditions: 10 mM HEPES-KOH, pH 8.2. Protein concentration was 5–23 µM. Excitation wavelength was 275 nm. The dashed curves are theoretical fits computed according to the sequential metal-binding scheme [IV] (see Table 4 and Table 5). (a) The dependence of Ocm fluorescence intensity at 306 nm on total concentration of Ca²⁺/EDTA during saturation of apo-Ocm with Ca²⁺, followed by EGTA-induced Ca²⁺ removal. (b) Ca²⁺ to protein molar was 2:1; the Ca²⁺ removal was initiated via addition of 1 mM EGTA.

Figure 5

Determination of Mg²⁺ affinity of rWT/C18S Ocm at 20 °C by equilibrium dialysis. Ocm concentration was 50–200 μM. Buffer conditions: 30 mM HEPES-KOH, 1 mM EGTA, pH 7.4 and 2–750 μM Mg(NO₃)₂. [bMg²⁺], concentration of Mg²⁺ bound to Ocm; [fMg²⁺], free Mg²⁺ concentration; P₀, total Ocm concentration. (a) Dependence of the number of Mg²⁺ ions bound per Ocm molecule upon pMg. The points are experimental, dashed curves are theoretical computed according to the sequential metal binding scheme [IV] using Equation (5) (see Table 4). (b) Scatchard plot.