Structures and metal-ion-binding properties of the Ca2+-binding helix–loop–helix EF-hand motifs

Gifford, Jessica L.; Walsh, Michael P.; Vogel, Hans J.

doi:10.1042/bj20070255

Cited by 787 publications

(1,044 citation statements)

References 178 publications

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“…EF-loops are defined here as the structural motifs present within the EF-hand domain responsible for Ca 2+ -binding. Paired EF-loops are usually only found in Ca 2+ -effector proteins (Gifford et al, 2007). Indeed, the sequences corresponding to these domains in both PLCη proteins are conserved relative to the EF-hands of other proteins.…”

Section: Analysis Of Ef-hand Domains From Plcη Enzymesmentioning

confidence: 99%

“…This is then translated, often through a complex of proteins, into a physiological event. Many of these Ca 2+ -effector proteins contain homologous Ca 2+ -binding domains termed EF-hands (Strynadka and James, 1989;Gifford et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…This structure is further stabilized by a short antiparallel β-sheet formed between the pairs' EF-loops (Grabarek, 2006). The structural and metal ion-binding properties of these domains are reviewed by Gifford et al (2007). Examples of Ca 2+ -responsive effector proteins that contain EF-hand domains include calmodulin (CaM; Vetter and Leclerc, 2003), neuronal calcium sensor-1 (NCS-1; Burgoyne, 2004) and troponin C (Parmacek and Leiden, 1991).…”

Section: Introductionmentioning

confidence: 99%

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A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

Popovics

Kamil

Morgan

et al. 2014

J of Cellular Biochemistry

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Phospholipase C-ɳ(PLCɳ) enzymes are a class of phosphatidylinositol 4,5-bisphosphatehydrolyzing enzymes involved in intracellular signaling. PLCɳ2 can sense Ca 2+ (stimulated bỹ 1 µM free Ca 2+ ) suggesting that it can amplify transient Ca 2+ signals. PLCɳ enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca 2+ -binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCɳ2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[ 1 H, 1 H] TOCSY NMR. Both PLCɳ2 EF-loop peptides bound Ca 2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCɳ2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca 2+ -binding. To determine whether the EF-hand is responsible for Ca 2+ -sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCɳ2 proteins. Addition of 70µM monensin (a Na + /H + antiporter that increases intracellular Ca 2+ ) induced a 4 to 7-fold increase in wild-type PLCɳ2 activity. In permeabilized cells, PLCn2 exhibited a ~4-fold increase in activity in the presence of 1 µM free Ca 2+ . The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca 2+ -sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca 2+ -sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca 2+ -sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca 2+ -sensing.

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Section: Analysis Of Ef-hand Domains From Plcη Enzymesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

Popovics

Kamil

Morgan

et al. 2014

J of Cellular Biochemistry

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“…All members of this family carry at least one pair of EF-hand calcium binding sites that bind calcium with equilibrium affinity constants ranging from 10 4 M in the case of S100 proteins, to 10 8 M as found for parvalbumin. 1 The protein affinity for Ca 21 is fine-tuned by the amino acid sequence of the EF-hand binding site, overall structure of the Ca 21 sensor, and by interactions with other intracellular partners. Despite relatively high sequence similarities, EF-hand proteins exhibit large structural diversity in the apo-and Ca 21 bound forms that is essential for specific interactions with intracellular partners and functional diversity.…”

Section: Introductionmentioning

confidence: 99%

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

et al. 2015

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Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca 21 concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca 21 /Mg 21 association to DREAM. Our data indicate a minor impact of Ca 21 association on the overall structure of the Nand C-terminal domains, although Ca 21 binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca 21 bound forms of the protein. Time-resolved fluorescence data indicate that Ca 21 binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca 21 association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca 21 and Mg 21 suggesting that, under physiological conditions, Ca 21 free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.

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“…The functional EF-hand motifs lie within residues 66-94 (EF-2), 102-130 (EF-3), and 147-175 (EF-4). In the three functional EFhands of NCS-1, conserved glutamic acidic residues at positions 84, 120, and 168 provide a bidentate ligand necessary to coordinate calcium [11]. Binding of calcium at these three sites induces conformational changes in the protein structure that expose a hydrophobic pocket that has been proposed to switch the protein to its active state [12][13].…”

Section: Introductionmentioning

confidence: 99%

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

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We have developed a protocol to produce large quantities of high purity myristoylated and nonmyristoylated Neuronal Calcium Sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6X His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100 -200 µM). With optimized growth conditions and a high capacity metal affinity purification scheme, > 50 mg of homogenous myristoylated NCS-1 is obtained from 1 L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 EF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.

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Structures and metal-ion-binding properties of the Ca2+-binding helix–loop–helix EF-hand motifs

Cited by 787 publications

References 178 publications

A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

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Structures and metal-ion-binding properties of the Ca2+-binding helix–loop–helix EF-hand motifs

Cited by 787 publications

References 178 publications

A Canonical EF‐Loop Directs Ca2+‐Sensitivity in Phospholipase C‐η2

A Canonical EF‐Loop Directs Ca2+‐Sensitivity in Phospholipase C‐η2

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

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A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator