The Engineering of Binding Affinity at Metal Ion Binding Sites for the Stabilization of Proteins: Subtilisin as a Test Case

abstract;A weak Ca²⁺binding site in the bacterial serine protease subtilisin BPN′ (EC 3.4.21.14) was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca²⁺binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN’ at 65 °C as a function of the free [Ca²⁺]. Increasing the [Ca²⁺] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca²⁺specific binding site with an apparent log K_a= 1.49. A series of refined X-ray crystal structures (R ≤ 0.15, 1.7 Å) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl₂has allowed a detailed structural characterization of this Ca²⁺binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca²⁺by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca²⁺binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 Å, respectively, from the bound Ca²⁺. These distances and the degree of enhanced binding are consistent with simple electrostatic theory. Moreover, when both Asp changes were introduced together, the binding affinity for Ca²⁺was found to be increased about 6-fold over that for the wild-type protein, suggesting an independent and nearly additive effect on the total electrostatic potential at this locus.