Effects of Myosin Light Chain Kinase and Peptides on Ca2+ Exchange with the N- and C-terminal Ca2+ Binding Sites of Calmodulin

Abstract

2؉dissociates 5-29 times faster from the N-terminal in these CaM⅐peptide complexes and both lobes are required for activation, Ca 2؉ dissociation from the N-terminal would control target protein inactivation. Ca

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

“…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.…”

“…2), where each dissociation event can be attributed to the essentially simultaneous dissociation of two Ca2+ ions from one domain of CaM (Suko et al, 1986;Martin et al, 1992;Johnson et al, 1996;Persechini et al, 1996). In the presence of the target peptides these events are slowed dramatically, with the highest Ca2+ affinity CaM-peptide complexes yielding the slowest Ca2+ dissociation rates.…”

“…For free CaM, it is well established that the N domain provides the low affinity, rapidly dissociating component, while the C domain is responsible for the high affinity, slowly dissociating component (Martin et al, 1985(Martin et al, , 1992Linse et al, 1991;Johnson et al, 1996). We have confirmed this assignment in our Drosophila CaM by stopped-flow absorbance measurements at 276 nm, where the Ca2+-dependent absorbance of Tyr-138 provides a specific probe of Ca2+ binding to EF-hand site IV in the C domain .…”

“…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).…”