![]() ![]() ![]() This fragment encompasses EF-2 and EF-3, as well as the intervening sequence that normally serves as the flexible linker between its N- and C-domains. To investigate further how EF-hand association affects the structure, Ca 2+ affinity, and cooperativity of CaM, we examined a previously uncharacterized fragment of this protein spanning residues 46–113 (CaM2/3) ( Fig. Heterodimers of EF-hand peptides have improved Ca 2+ affinities over those of homodimers, yet are still significantly lower than in their respective native proteins ( Finn et al. Despite this structural similarity, Ca 2+ binding and cooperativity are compromised in the homodimeric EF-hand models ( Reid 1987b Kay et al. The tertiary structures of the two TnC homodimers are very similar to the C-domain of native TnC and, by extension, all EF-hand domains ( Kay et al. Heterodimers include EF-1:EF-2 of CaM or calbindin D9k and EF-3:EF-4 of CaM or TnC ( Finn et al. 1992), and chimeras of parvalbumin and CaM ( Franchini and Reid 1999). Examples of homodimers include single EF-hand peptides of CaM (EF-3) ( Reid 1987a), TnC (EF-3 and EF-4) ( Kay et al. This is evidenced by the peptide models of single EF-hands that form heterodimers, as well as those that self-associate as homodimers. This optimization includes the ability to cooperatively bind Ca 2+ with an overall affinity necessary to respond to changes in the physiological concentrations of this cation and thereby regulate numerous signaling pathways.Īlthough EF-hand sequences have evolved to specifically pair into domains, there is still remarkable plasticity in their potential modes of association. Accordingly, the “odd–even” paired EF-1 and EF-2 have evolved to optimize their association to form the N-domain of CaM, as have EF-3 and EF-4 to form the C-domain. This in turn duplicated into a two-domain progenitor EF-hand protein, which finally diverged into a family of Ca 2+-binding proteins, including CaM and troponin C (TnC) ( Nakayama et al. In this theory, a progenitor EF-hand gave rise to a progenitor EF-hand domain. The arrangement of EF-hands in CaM is thought to be a consequence of its evolution from a progenitor EF-hand by gene duplication. The two domains are tethered through a flexible intervening sequence, known as the linker ( Babu et al. As a prototypical EF-hand protein, calmodulin (CaM) contains four Ca 2+ binding EF-hand motifs: EF-1 and EF-2 associate to form its N-domain, and EF-3 and EF-4 associate to form its C-domain. A key feature of this hydrophobic packing is the presence of four stacked aromatic residues found on the first and the fourth helices of the domain ( Babu et al. An EF-hand domain, which is typically composed of two EF-hands connected by a flexible loop, is stabilized by an antiparallel β-sheet and by the packing of hydrophobic side chains from neighboring helices. This nonnative hydrophobic core packing may contribute to the weak Ca 2+ affinity exhibited by EF-2 in the context of CaM2/3.Ĭalcium (Ca 2+) binding proteins are often composed of EF-hand motifs associated into folded EF-hand domains. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its “even–odd” paired EF-hands. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. Binding of the first equivalent of Ca 2+ induces the cooperative folding of CaM2/3. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca 2+ binding with a K d1 = 30 ± 5 μM to EF-3, and a K d2 > 1000 μM to EF-2. Despite having an “even–odd” pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the “odd–even” paired N- and C-domains of Ca 2+-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. Based on NMR spectroscopic analyses, Ca 2+-free CaM2/3 is predominantly unfolded, but upon binding Ca 2+, adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel β-sheet. In this study, we examined the structure, dynamics, and Ca 2+-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca 2+)-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). ![]()
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