TUNING THE EQUILIBRIUM ION AFFINITY AND SELECTIVITY OF THE EF-HAND CALCIUM-BINDING MOTIF - SUBSTITUTIONS AT THE GATEWAY POSITION

The ion binding parameters of the EF-hand Ca2+ binding motif are caref ully tuned for different biological applications. The present study ex amines the contribution of the ninth position of the Ca2+ coordinating EF-loop to the tuning of Ca2+ affinity and selectivity, using the mod el EF-loop of the Escherichia coli galactose binding protein. Eight si de chains, representing the entire set of side chains commonly observe d in natural EF-loop sequences, are tested at the ninth position of th e 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 sel ectivities of the engineered sites are quantitated. Neutral side chain s of different size at the ninth EF-loop position [Gin (wild type), As n, Thr, Ser, Ala, Gly] are observed to yield similar Ca2+ affinities a nd retain the native ability to exclude the physiological competing me tal cations Na+, K+, and Mg2+. Acidic gateway side chains (Glu, Asp) a re found to reduce Ca2+ affinity and shift the ionic charge selectivit y as much as 10(3)-fold toward trivalent cations. Relative to the nati ve Gin, all engineered side chains cause a partial loss of ionic size selectivity, stemming from enhanced affinities for nonphysiological la rge ions. Overall, the results have implications for the molecular mec hanisms used by the EF-loop to control both (i) charge selectivity, wh ich is proposed to stem from the electrostatic repulsion between the c oordinating 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 TD3+ binding and r elease without shifting the equilibrium affinity of this ion [Drake, S . K., & Falke, J. J. (1996) Biochemistry 35, 1753-1760]. The present r esults 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.