Target recognition by calmodulin: Dissecting the kinetics and affinity of interaction using short peptide sequences

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N‐terminal target residues and the C‐domain of calmodulin, and between the C‐terminal target residues and the N‐domain of calmodulin (Ikura M et al., 1992, Science 256:632–638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C‐domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1–10 of M13) binds to CaM with Kd ≈︁ 1 μM; peptide FW10 (residues 9–18 of M13, with Phe‐17 → Trp substitution) binds to CaM with Kd ≈︁ 100 μM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C‐domain, forming a stable half‐saturated complex, Ca2‐CaM‐peptide, and confirming the functional importance of the interaction of this sequence with the C‐domain. Stopped‐flow studies show that the EGTA‐induced dissociation of WF10 from Ca4‐CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half‐saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N‐terminal target residues with the C‐domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N‐ and C‐domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin‐dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C‐domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.

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