Sk. Drake et al., TUNING THE EQUILIBRIUM ION AFFINITY AND SELECTIVITY OF THE EF-HAND CALCIUM-BINDING MOTIF - SUBSTITUTIONS AT THE GATEWAY POSITION, Biochemistry, 35(21), 1996, pp. 6697-6705
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.