The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

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 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.