| Summary: | Several studies have revealed that calcium-binding EF-hands associate in various arrangements resulting in unique calcium affinities and target interactions, yet similar structures. This study examines the effect of EF-hand association on the structure, calcium-binding, and target-enzyme activation of a novel fragment of calmodulin formed by the association of EF-hands 2 and 3 (CaM2/3). The results from CaM2/3 are compared to those for the N- and C-domain fragments of calmodulin (CaM1/2 and CaM3/4, respectively). Based on NMR spectroscopic analyses, CaM2/3 is unstructured in the absence of calcium, but upon binding calcium adopts a structure that is similar to both the N- and C-domains of calmodulin. CaM2/3 is composed of four α-helices and a short anti-parallel β-sheet between EF-hands 2 and 3. These EF-hands associate in a manner similar to those in the N- and C-domains of calmodulin. This association is facilitated by the conformationally-flexible hinge region between EF-hands. CaM2/3 exhibits stepwise calcium-binding with a Kd₁ = 30 ± 5 μM to EF-hand 3, and a Kd₂ = 1800 ± 400 μM to EF-hand 2. In native calmodulin, aromatic residues opposite the loop between EF-hands are an important part of its hydrophobic core. It is suggested here that these residues act as an "aromatic zipper" to hold the first and fourth helices of each domain together. In CaM2/3 this "aromatic zipper" is in a structurally redundant position adjacent to the loop between EF-hands, and this may result in weak calcium-binding affinity at EF-hand 2. Our studies reveal that CaM2/3 binds a peptide corresponding to the calmodulin-binding sequence on skeletal muscle myosin light chain kinase (M13). This affinity is lower than that measured for both CaM1/2 and CaM3/4. MI3 appears to bind to a hydrophobic binding pocket on CaM2/3 in two equally populated conformations. CaM1/2 and CaM3/4 have little or no ability to stimulate the activity of calcineurin. However, CaM2/3 has a limited ability to stimulate calcineurin, but also appears to inhibit the stimulatory effect of calmodulin on calcineurin. This investigation leads to a greater understanding of the effect of EF-hand pairing, on structure, calcium-binding affinity, and target interactions in calmodulin. === Pharmaceutical Sciences, Faculty of === Graduate
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