Amino Acid Contributions to Binding across Paired EF-Hands in Calmodulin

Abstract

The ubiquitous calcium-binding protein calmodulin participates in a network of interactions influencing cellular processes like gene regulation and ion channel modulation. The immediate function of calmodulin is to bind calcium to its four EF-hand calcium binding domains grouped into two pairs, each pair residing in either lobe of the dumbbell-shaped protein. Ligand binding involves a constellation of amino acid interactions localized within the binding site as well as across paired sites. Our previous work has shown that each of the four binding sites has a unique apparent affinity that appears to have been selected for during evolution. We have investigated how the differences in binding loop amino acid sequences cause differences in binding affinity of the individual EF-hands, as well as how the amino acid sequence of one site influences binding at its paired site through cooperative mechanisms. Between the two paired N-lobe EF-hands, all but five positions within twelve residue binding loops are identical. Each of these five loop positions were individually mutated to its counterpart from the paired site and site-specific binding measurements were performed on N-lobe fragments to explore the two roles each position plays to determine binding in the paired EF-hand system: locally within their own site (cis-effects) and at the paired site (trans-effects). Several mutations had both cis- and trans- effects whereas others had predominantly cis- or trans- effects. The complexity of the mutant effects on binding of both sites suggests that the N-lobe has been conserved to act as a complex multiple site calcium binding unit, rather than two independent EF-hands.