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

Abstract

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 256632-638). 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 WFlO (residues 1-10 of M13) binds to CaM with Kd = 1 pM; peptide FWlO (residues 9-18 of M13, with Phe-17 -+ Trp substitution) binds to CaM with Kd = 100 pM. The effect of peptide WFlO 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 halfsaturated 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 WFlO 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.

Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+ binding and implications for kinase activation

“…Figure 1 directly compares the Ca2+ binding profiles of CaM alone and in the presence of the five full-length peptides. The Ca2+ concentrations yielding half-saturation ([Ca2+],,2), summarized in Table 2, reveal that target peptides increase the macroscopic Ca2+ affinity of CaM by as much as two orders of magnitude, as previously shown in similar peptide studies using a variety of experimental conditions (Yazawa et al, 1992;Stemmer & Klee, 1994;Bayley et al, 1996). Here, the controlled conditions enable direct comparison of intermolecular tuning by the five different peptides.…”

“…Previous studies have characterized the effects of individual target peptides on Ca2+ binding to CaM (Vorherr et al, 1990;Yazawa et al ., 1992;Bayley et al, 1996) or have compared the effects of multiple target sequences on Ca2+ dissociation kinetics (Johnson et al, 1996;Persechini et al, 1996). The present work extends these studies by systematically comparing both the equilibrium and kinetic aspects of Ca2+ binding to nine CaM-peptide complexes and one CaM-protein complex.…”

“…This approach is justified by observations that the Ca2+ binding profile of free CaM can be reconstructed from those of its two isolated domains, by evidence for domain-independence in the binding and dissociation of Ca2+ in CaM-target complexes, and by the observation that individual Ca2+ site mutations perturb mainly the two sites in the same domain (Linse et al, 1991;Starovasnik et al, 1992;Finn et al, 1995;Bayley et al, 1996;Johnson et al, 1996). Table 2 presents the resulting equilibrium dissociation constants and Hill coefficients of the high-affinity ( K D l , H I ) and low affinity (KD2, Hz) CaM domains.…”

“…Synthetic peptides corresponding to the CaM binding sequences of specific proteins serve as potent antagonists in Ca2+/CaM-dependent activation assays and bind CaM with nanomolar affinities that are comparable to or tighter than those of the full-length proteins Lukas et al, 1986;Dasgupta et al, 1989;Anagli et al, 1995). Peptides can also increase the Ca2+ affinity and slow Ca2+ dissociation rates as compared to CaM in the absence of a target sequence (Yazawa et al, 1992;Stemmer & Klee, 1994;Bayley et al, 1996;Johnson et al, 1996;Persechini et al, 1996).…”

Rational Design of New Binding Specificity by Simultaneous Mutagenesis of Calmodulin and a Target Peptide

“…Many studies have probed the contributions of both the charged and hydrophobic anchor residues to CaM binding affinity, (5,6,(19)(20)(21)(22) but relatively few have sought to manipulate specificity through purposeful mutagenesis of one or both binding partners. A step in this direction was taken by Mayo and colleagues, (23,24) and involved the integration of computational and experimental methods to bias CaM specificity toward one of its many target peptides.…”