Calmodulin activates the skeletal muscle Ca 2؉ release channel RYR1 at nM Ca 2؉ concentrations and inhibits the channel at M Ca 2؉ concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nM and M Ca 2؉ concentrations and are required for maximum inhibition of the channel at M Ca 2؉ concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nM and M Ca 2؉ concentrations, but destroyed its functional effects on RYR1 at nM Ca 2؉ . Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca 2؉ -calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca 2؉ binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the Nterminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.
Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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 alpha-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 ((Ca(2+))(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 (PCaM(1-80) and PCaM(76-148)), as well as with the C-domain of full-length PCaM (PCaM(1-148)). In the presence of calcium, all of these interactions were again observed, in addition to which an association with the N-domain of (Ca(2+))(4)-PCaM(1-148) 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.
Membrane-associated guanylate kinases (MAGUKs) regulate the formation and function of molecular assemblies at specialized regions of the membrane. Allosteric regulation of an intramolecular interaction between the Src homology 3 (SH3) and guanylate kinase (GK) domains of MAGUKs is thought to play a central role in regulating MAGUK function. Here we show that a mutant of the Drosophila MAGUK Discs large (Dlg), dlg sw , encodes a form of Dlg that disrupts the intramolecular association while leaving the SH3 and GK domains intact, providing an excellent model system to assess the role of the SH3-GK intramolecular interaction in MAGUK function. Analysis of asymmetric cell division of maternal-zygotic dlg sw embryonic neuroblasts demonstrates that the intramolecular interaction is not required for Dlg localization but is necessary for cell fate determinant segregation to the basal cortex and mitotic spindle alignment with the cortical polarity axis. These defects ultimately result in improper patterning of the embryonic central nervous system. Furthermore, we demonstrate that the sw mutation of Dlg results in unregulated complex assembly as assessed by GukHolder association with the SH3-GK versus PDZ-SH3-GK modules of Dlg sw . From these studies, we conclude that allosteric regulation of the SH3-GK intramolecular interaction is required for regulation of MAGUK function in asymmetric cell division, possibly through regulation of complex assembly.The membrane-associated guanylate kinase (MAGUK) 2 superfamily consists of ubiquitous scaffolding proteins that are composed of a common core of contiguously linked modular domains (protein-protein interaction domains, PDZ and SH3 domains, and a domain with homology to the yeast guanylate kinase, GK domain). MAGUKs are concentrated at sites of cellcell contact (1) and organize a variety of cell adhesion molecules, cytoskeletal proteins, receptors, ion channels, and associated signaling molecules at specialized regions of the membrane (2). Protein complex organization by MAGUKs has been thought to occur, at least in part, through allosteric regulation that arises from an intramolecular interaction between the SH3 and GK domains. This interaction has been shown to regulate binding of numerous MAGUK ligands in vitro (3-5). However, whereas the SH3-GK interaction has been extensively characterized biochemically (6 -8), very little data exists regarding its physiological relevance. Here we investigate the role of the SH3-GK intramolecular interaction of Discs large (Dlg), a prototype of the MAGUK superfamily.Loss of dlg activity results in overgrowth of imaginal discs and tumor formation (9). Dlg localizes to septate and neuromuscular junctions and is essential for establishing and maintaining apicobasal polarity of Drosophila epithelia (10). Dlg also plays an important role in regulating the process of asymmetric cell division (ACD) (11-13). ACD is a mechanism for generating cellular diversity via unequal mitotic divisions of progenitor cells. For instance, in wild-type Drosophila...
Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.
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