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 con served in sequence. Members of this family include parvalbumin and many pro minent regulatory proteins such as calmodulin and troponin C. EF-hand prote ins are involved in a variety of physiological processes including cell-cyc le 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 lo op. An E101D substitution has been made in the parvalbumin EF site. The sub stitution decreases the Ca2+-binding affinity 100-fold and increases the Mg 2+-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 geome try 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 t he sevenfold coordination preferred by Ca2+, presumably because of strain l imits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the cha racteristics of the last coordinating residue and the plasticity of the Ca2 +-binding loop delimit the allowable geometries for the coordinating sphere .