Molecular mechanism of the Ca2+-dependent activation of sorcin (soluble resistance-related calcium binding protein. A study with site-specific mutants

Sorcin is a two-domain protein belonging to the penta-EF-hand family that traslocates reversibly from cytosol to membranes through a Ca2+-dependent interaction with protein targets. Although EF1, EF2, EF3 are potentially able to bind calcium at micromolar concentrations, binding of two Ca2+/monomer activates sorcin and triggers translocation. To identify the functional pair, the conserved bidentate –Z glutamate in these EF-hands was mutated to yield E53Q-, E94A-, E124A-sorcin, respectively. The behavior of the three sitespecific sorcin mutants shows that the EF3 hand is the site endowed with the highest affinity for calcium and that EF2 and EF3 that are not paired structurally are the functional EF-hand pair. Information of Ca2+ binding to EF3 was proposed to be transferred to the rest of the molecule by means of the long and rigid D-helix that is shared by EF2 and EF3. To establish whether this helix is instrumental in sorcin activation, two D-helix residues were mutated into glycine: W105 involved in the network of interaction around the helix itself, and W99, which faces solvent. The substitution of W105 almost abolishes the capacity of sorcin to interact with its molecular targets while mutation of W99 has little effect. Disruption of the interaction network around the D-helix, therefore, inhibits information transfer from the EF3 hand demonstrating the central role of the D-helix in the Ca2+-dependent activation of sorcin