Tuning the Equilibrium Ion Affinity and Selectivity of the EF-Hand Calcium Binding Motif: Substitutions at the Gateway Position

Abstract

The ion binding parameters of the EF-hand Ca²⁺ binding motif are carefully tuned for different biological applications. The present study examines the contribution of the ninth position of the Ca²⁺-coordinating EF-loop to the tuning of Ca²⁺ affinity and selectivity, using the model EF-loop of the Escherichia coli galactose binding protein. Eight side chains, representing the entire set of side chains commonly observed in natural EF-loop sequences, are tested at the ninth position of the model EF-loop to determine their effects on equilibrium ion binding parameters. Using the spherical metal ions of groups Ia, IIa, and IIIa and the lanthanides as probes, both the Ca²⁺ affinities and ionic selectivities of the engineered sites are quantitated. Neutral side chains of different size at the ninth EF-loop position [Gln (wild type), Asn, Thr, Ser, Ala, Gly] are observed to yield similar Ca²⁺ affinities and retain the native ability to exclude the physiological competing metal cations Na⁺, K⁺, and Mg²⁺. Acidic gateway side chains (Glu, Asp) are found to reduce Ca²⁺ affinity and shift the ionic charge selectivity as much as 10³-fold toward trivalent cations. Relative to the native Gln, all engineered side chains cause a partial loss of ionic size selectivity, stemming from enhanced affinities for nonphysiological large ions. Overall, the results have implications for the molecular mechanisms used by the EF-loop to control both (i) charge selectivity, which is proposed to stem from the electrostatic repulsion between the coordinating oxygens, and (ii) size selectivity, which is theorized to involve complex interactions between multiple coordinating side chains. Finally, it has recently been shown that the ninth EF-loop position serves as a “gateway” to modulate the kinetics of Tb³⁺ binding and release without shifting the equilibrium affinity of this ion [Drake, S. K., & Falke, J. J. (1996) Biochemistry 35, 1753−1760]. The present results confirm that isoelectric substitutions at the gateway position have little effect on Ca²⁺ affinity, thereby supporting the hypothesis that the gateway side chain provides kinetic tuning of Ca²⁺ signaling proteins independently of their Ca²⁺ activation thresholds.