Harnessing Environmental Ca for Extracellular Protein Thermostabilization.

Ca is the third-most prevalent metal ion in the environment. EF hands are common Ca-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca concentration fluctuations by coupling Ca binding to changes in protein structure. We show that an extracellular structural Ca-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca to the EF hand of the extracellular (periplasmic) glucose-galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca-mediated stability. A disorder → order transition accompanies the formation of the Ca complex in the folded state and dictates the minimum Ca concentration at which the Ca-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca-mediated control elements in signal transduction pathways. Ca-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.