Showing papers by "Madeline A. Shea published in 2008"
TL;DR: It is shown that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM and highlights the critical importance of oxidation-dependent CaMK II activation to AngII and ischemic myocardial apoptosis.
989 citations
TL;DR: CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium, which contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C- domain of CaM.
Abstract: Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901−1938) [Mori, M., et al. (2000) Biochemistry 39, 1316−1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ pepti...
55 citations
TL;DR: The use of fluorescence and NMR spectroscopy has been used to determine the free energies of calcium binding to the PCaM‐melittin complex, and to estimate interdomain cooperativity, which supports a model that may apply more generally to ion channels that associate with the C‐domain of CaM under low (resting) calcium conditions, but rearrange when calcium binding triggers an association of the N‐ domain with the channel.
Abstract: Calmodulin (CaM) is the primary transducer of calcium fluxes in eukaryotic cells. Its two domains allosterically regulate myriad target proteins through calcium-linked association and conformational change. Many of these proteins have a basic amphipathic α-helix (BAA) motif that binds one or both CaM domains. Previously, we demonstrated domain-specific binding of melittin, a model BAA peptide, to Paramecium CaM (PCaM): C-domain mutations altered the interaction with melittin, whereas N-domain mutations had no discernable effect. Here, we report on the use of fluorescence and NMR spectroscopy to measure the domain-specific association of melittin with calcium-saturated ((Ca2+)4-PCaM) or calcium-depleted (apo) PCaM, which has enabled us to determine the free energies of calcium binding to the PCaM-melittin complex, and to estimate interdomain cooperativity. Under apo conditions, melittin associated with each PCaM domain fragment (PCaM1–80 and PCaM76–148), as well as with the C-domain of full-length PCaM (PCaM1–148). In the presence of calcium, all of these interactions were again observed, in addition to which an association with the N-domain of (Ca2+)4-PCaM1–148 occurred. This new association was made possible by the fact that melittin changed the calcium-binding preferences for the domains from sequential (C > N) to concomitant, decreasing the median ligand activity of calcium toward the N-domain 10-fold more than that observed for the C-domain. This selectivity may be explained by a free energy of cooperativity of −3 kcal/mol between the N- and C-domains. This study demonstrates multiple domain-selective differences in the interactions between melittin and PCaM. Our findings support a model that may apply more generally to ion channels that associate with the C-domain of CaM under low (resting) calcium conditions, but rearrange when calcium binding triggers an association of the N- domain with the channel. Proteins 2008. © 2008 Wiley-Liss, Inc.
38 citations