Equilibrium Unfolding of Neuronal Calcium Sensor-1

Dasari, Muralidhar; Jobby, Maroor K.; Krishnan, Kannan; Annapurna, V.; Chary, Kandala V. R.; Jeromin, Andreas; Sharma, Yogendra

doi:10.1074/jbc.m414243200

Cited by 31 publications

(9 citation statements)

References 31 publications

(33 reference statements)

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“…The Ca 2ϩ -induced structural change to EF-SAM translates into a 3.2-kcal mol Ϫ1 augmentation in thermodynamic stability. This extrinsic Ca 2ϩ stabilization is in the same range as other EF-hand-containing proteins such as neuronal calcium sensor-1 (ϩ3.5 kcal mol Ϫ1 ), tryptic C-terminal fragment of calmodulin (ϩ3.0 kcal mol Ϫ1 ), and tryptic N-terminal fragment of calmodulin (ϩ3.8 kcal mol Ϫ1 ) (50,51). The conformational changes accompanying Ca 2ϩ depletion promote a monomer-to-oligomer transition for EF-SAM.…”

Section: Discussionmentioning

confidence: 65%

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Stathopulos

Plevin

et al. 2006

Journal of Biological Chemistry

364

398

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-loaded EF-SAM (holo) contains high ␣-helicity, whereas EF-SAM in the absence of Ca 2؉ (apo) is much less compact. Accordingly, the melting temperature (T m ) of the holoform is ϳ25°C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca 2؉ -induced stabilization of 3.2 kcal mol ؊1 . We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca 2؉ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (K d ) of ϳ0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca 2؉ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca 2؉ sensor.Calcium is a fundamental signaling messenger in every eukaryotic cell, regulating a multitude of diverse and kinetically distinct cellular phenomena including gene transcription, protein folding, protein degradation, apoptosis, necrosis, and exocytosis, to name a few (1). The endoplasmic reticulum (ER) 4 is a network of folded membranes that extends through the cytoplasm to the nuclear envelope of eukaryotes. The ER membranes surround an inner cavity, the lumen, that is critical to the function of the ER as a Ca 2ϩ signaling organelle (2). Because vital Ca 2ϩ -dependent processes are associated with the ER, it is essential that changes in luminal Ca 2ϩ levels do not adversely affect these phenomena. Eukaryotes have evolved store-operated Ca 2ϩ entry (SOCE), also termed capacitive Ca 2ϩ entry, as a major Ca 2ϩ entry pathway in electrochemically non-excitable cells (3-5). SOCE is the process whereby modest ER Ca 2ϩ store depletion leads to plasma membrane (PM) Ca 2ϩ release-activated channel (CRAC) activation, providing a sustained Ca 2ϩ elevation in the cytoplasm from extracellular sources and ultimately refilling the ER luminal Ca 2ϩ stores (6). Until recently, the molecular link between ER Ca 2ϩ efflux and extracellular influx was not known. However, interfering and small inhibiting RNA studies have independently implicated stromal interaction molecule-1 (STIM1) as the likely Ca 2ϩ sensor in the ER (7,8). This single-pass, type I transmembrane protein of 685 amino acids has been found localized on both the plasma and ER membranes (3-5). The N-terminal regions of STIM1 include a signal peptide, putative EF-hand motif, and predicted sterile ␣-motif (SAM) domain. The cytosolic C-terminal region consists of two coiled-coil domains, a Pro/Ser-rich region, and a Lys-rich region (supplemental Fig. 1A) (9 -11). The putative EF-hand of STIM1 strongly aligns with the helix-loop-helix consensus sequence of this Ca 2ϩ binding motif (supplemental Fig. 1B). The EF-hand in STIM1 is somewhat unorthodox because it is seemingly unpaired, whereas Ca 2ϩ sensor pr...

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Section: Discussionmentioning

confidence: 65%

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Stathopulos

Plevin

et al. 2006

Journal of Biological Chemistry

364

398

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“…To define the relative contributions of these EF-hands, we constructed and characterized, using a FA assay, variants of NCS-1 that prevented Ca 2+ binding at specific sites (E84Q disrupts EF-2, E120Q disrupts EF-3, E168Q disrupts EF-4, and ΔEF contains three point mutations to disrupt all functional EF-hands). These were chosen because they disrupt Ca 2+ binding without significantly affecting NCS-1 structure (19, 32, 33). …”

Section: Resultsmentioning

confidence: 99%

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

et al. 2011

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Neuronal calcium sensor-1 (NCS-1) is a small calcium binding protein that plays a key role in the internalization and desensitization of activated D2 dopamine receptors (D2Rs). Here, we have used fluorescence anisotropy (FA) and a panel of NCS-1 EF hand variants to interrogate the interaction between the D2R and NCS-1. Our data is consistent with the following conclusions: 1) FA titration experiments indicate that at low D2R peptide concentrations calcium-loaded NCS-1 binds to the D2R peptide in a monomeric form. At high D2R peptide concentrations, the FA titration data is best fit by a model in which the D2R peptide binds two NCS-1 monomers sequentially in a cooperative fashion. 2) Competition FA experiments in which unlabeled D2R peptide was used to compete with labeled peptide for binding to NCS-1 shifted titration curves to higher NCS-1 concentrations, suggesting that the binding of NCS-1 to the D2R is highly specific and that binding occurs in a cooperative fashion. 3) N-terminally myristoylated NCS-1 dimerizes in a calcium-dependent manner. 4) Co-immunoprecipitation experiments in HEK293 confirm that NCS-1 can oligomerize in cell lysates, and that oligomerization is dependent on calcium binding and requires functionally intact EF hand domains. 5) Ca2+/Mg2+ FA titration experiments revealed that NCS-1 EF-hands 2-4 contributed to binding with the D2R peptide. EF-2 appears to have the highest affinity for Ca2+ and occupancy of this site is sufficient to promote high-affinity binding of the NCS-1 monomer to the D2R peptide. Magnesium ions may serve as a physiological co-factor with calcium for NCS-1/D2R binding. Finally, we propose a structural model that predicts that the D2R peptide binds to the first 60 residues of NCS-1. Together, our results support the possibility of using FA to screen for small molecule drugs that can specifically block the interaction between the D2R and NCS-1.

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“…3, S2A,B, Table 1). All variants maintained a 3-state unfolding reaction as described earlier for the FL variant [37,45,46], with an intermediate state consisting of an unfolded N-domain and folded C-domain ( Fig. 3a) [36].…”

Section: A C-terminal Tail Is Vital For the Stability Of N-domainmentioning

confidence: 86%

Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

Staby

Kemplen

Stein

et al. 2020

Cell. Mol. Life Sci.

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Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

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Equilibrium Unfolding of Neuronal Calcium Sensor-1

Cited by 31 publications

References 31 publications

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Interaction between the D2 Dopamine Receptor and Neuronal Calcium Sensor-1 Analyzed by Fluorescence Anisotropy

Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

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