Metal-ion affinity and specificity in EF-hand proteins: coordination geometry and domain plasticity in parvalbumin

Background: The EF-hand family is a large set of Ca2+-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, miorotubule organization and vision. Results: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca2+-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca2+-binding affinity 100-fold and increases the Mg2+-binding affinity 10-fold. Both the Ca2+- and Mg2+-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. Conclusions: The E101D mutation does not affect the Mg2+ coordination geometry of the binding loop, but it does pull the F helix 1.1 Angstrom towards the loop. The E101D-Ca2+ structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca2+, presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca2+-binding loop delimit the allowable geometries for the coordinating sphere.