Browsing by Subject "Calmodulin"
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Item Open Access A maximum entropy-based approach for the description of the conformational ensemble of calmodulin from paramagnetic NMR(2016-05-04) Thelot, FrancoisCharacterizing protein dynamics is an essential step towards a better understanding of protein function. Experimentally, we can access information about protein dynamics from paramagnetic NMR data such as pseudocontact shifts, which integrate ensemble-averaged information about the motion of proteins. In this report, we recognize that the relative position of the two domains of calmodulin can be represented as the evolution of one of the domains in the space of Euclidean motions. From this perspective, we suggest a maximum entropy-based approach for finding a probability distribution on SE(3) satisfying experimental NMR measurements. While sampling of SE(3) is performed with the ensemble generator EOM, the proposed framework can be extended to uniform sampling of the space of Euclidean motions. At the end of this study, we find that the most represented protein conformations for calmodulin corresponds to conformations in which both protein domains are in close contact, despite being largely different from each other. Such a representation agrees with the random coil linker model, and sharply differs with the extended crystal structure of calmodulin.Item Open Access A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.(Nature, 2008-02-14) Dick, Ivy E; Tadross, Michael R; Liang, Haoya; Tay, Lai Hock; Yang, Wanjun; Yue, David TCa2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.Item Open Access Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.(EMBO J, 2010-09-01) Grötsch, Helga; Giblin, Jonathan P; Idrissi, Fatima-Zahra; Fernández-Golbano, Isabel-María; Collette, John R; Newpher, Thomas M; Robles, Virginia; Lemmon, Sandra K; Geli, María-IsabelMyosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.Item Open Access Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.(Biochemistry, 2010-05-18) Colomer, Josep; Schmitt, Allison A; Toone, Eric J; Means, Anthony RWe developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.Item Open Access Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel.(Cell, 2008-06-27) Tadross, Michael R; Dick, Ivy E; Yue, David TCalmodulin (CaM) in complex with Ca(2+) channels constitutes a prototype for Ca(2+) sensors that are intimately colocalized with Ca(2+) sources. The C-lobe of CaM senses local, large Ca(2+) oscillations due to Ca(2+) influx from the host channel, and the N-lobe senses global, albeit diminutive Ca(2+) changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca(2+) sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca(2+) oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca(2+) release from CaM combined with greater affinity of the channel for Ca(2+)-free versus Ca(2+)-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca(2+) sources.Item Open Access Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Ca(v)1.3 channels.(J Gen Physiol, 2010-03) Tadross, Michael R; Ben Johny, Manu; Yue, David TCa(2+)/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca(2+) channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within Ca(V)1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a "hinged lid-shield" mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a "shield" in Ca(V)1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca(2+) channelopathies involving S6 mutations.Item Open Access Regulation of CaMKKβ Dependent Signaling Pathways(2011) Green, Michelle FrancesCa2+/Calmodulin-dependent protein kinase kinase β(CaMKKβ) is a serine/threonine directed kinase which is activated following increases in intracellular Ca2+. CaMKKβ activates Ca2+/Calmodulin-dependent protein kinase I (CaMKI), Ca2+/Calmodulin-dependent protein kinase IV (CaMKIV), and the AMP-dependent protein kinase (AMPK) in a number of physiological pathways including learning and memory formation, neuronal differentiation, and regulation of energy balance. The purpose of the work presented in this dissertation is to better understand the regulation of CaMKKβ activity and specificity in CaMKKβ-dependent signaling cascades. First, the CaMKKβ-AMPK signaling complex is examined using biochemical assays. In both brain and cell lysates CaMKKβ and AMPK form a stable complex which can be examined by co-immunoprecipitation. This complex lacks the AMPKγ subunit and is not allosterically activated by adenosine 5'-monophohphate (AMP) binding. Using a series of CaMKKβ and AMPK mutants it was determined that the kinase domains of CaMKKβ and AMPK are necessary for their interaction and CaMKKβ must be active and bound to adenosine 5'-triphosphate (ATP) to form a complex with AMPK. However, CaMKKβ need not be active or bound to ATP to bind CaMKIV. This illustrates that the CaMKKβ-AMPK signaling complex differs from the CaMKKβ-CaMKIV signaling complex. These observations indicate that the CaMKKβ-AMPK signaling complex could be specifically targeted without effecting CaMKKβ-CaMKIV signaling.
Second, the regulation of CaMKKβ by multi-site phosphorylation is examined. Three phosphorylation sites in the N-terminus of CaMKKβ were identified by mass spectrometry which regulates its Ca2+/CaM-independent autonomous activity. The kinases responsible for these phosphorylations are identified as CDK5 and GSK3. These phosphorylation events are sequential with CDK5 priming for subsequent GSK3 phosphorylation. In addition to regulation of autonomous activity, phosphorylation of CaMKKβ regulates its half-life as determined in a radioactive pulse-chase assay. Examination of CaMKKβ in a cerebellar granule neuron model system demonstrates that CaMKKβ levels correlate with CDK5 activity and are regulated developmentally. In addition, appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.
Overall the work presented in this dissertation illustrates additional levels of regulation of CaMKKβ-dependent signaling pathways. In the future, these novel methods of CaMKKβ regulation will need to be considered when studying CaMKKβ-dependent signaling pathways.