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 10(3)-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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Drake SK,Lee KL,Falke JJdoi
10.1021/bi952430lsubject
Has Abstractpub_date
1996-05-28 00:00:00pages
6697-705issue
21eissn
0006-2960issn
1520-4995pii
bi952430ljournal_volume
35pub_type
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