Lanthanides as probes for calcium in biological systems

Martin, R. Bruce; Richardson, F. S.

doi:10.1017/s0033583500002754

Cited by 362 publications

(186 citation statements)

References 109 publications

(90 reference statements)

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“…However, even concentrations as low as 10 μM inhibited crystal formation. Lanthanides are often used to define calcium-binding sites in proteins (38); consequently, samarium was tested as a heavy atom derivative for the structure solution. A samarium-derivatized crystal (MCA2 C213G -Sm) was obtained that revealed a single, fully occupied Sm 3+ -binding site on the surface of the molecule (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of a Trypanosoma brucei metacaspase

McLuskey

Rudolf

Proto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

110

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Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded β-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.apoptosis | clan CD | parasite | X-ray crystallography P rogrammed cell death (PCD) is essential for animal development and the maintenance of adult tissues. PCD itself is a regulated process, and in animals, apoptosis is controlled through the action of caspases, aspartic acid-specific cysteine peptidases (1). PCD is also essential for plant development (2), and multiple markers for apoptosis have been described in yeast and a broad range of protozoan parasites (3), yet these organisms do not encode caspases in their genomes; thus, alternative pathways must have evolved for caspase-independent cell death to be regulated. In recent years, attention has focused on the metacaspases, which are a highly conserved group of caspase-like cysteine peptidases found in plants, fungi, and protozoa but not in the metazoa (4). In plants, metacaspases are essential for embryogenesis (5, 6) and have been shown to act in antagonistic relationships, functioning as both positive and negative regulators of PCD (7). In yeast and some protozoa, metacaspases have been implicated as mediators of cell death in response to oxidative stress and environmental change (3,(8)(9)(10).Various attempts have been made to draw parallels between caspases and metacaspases in respect to structure, activation, and function (11). However, two striking differences between metacaspases and caspases are their substrate specificities and requirements for activation. Metacaspases have arginine/lysine specificity, do not necessarily require proces...

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Section: Resultsmentioning

confidence: 99%

Crystal structure of a Trypanosoma brucei metacaspase

McLuskey

Rudolf

Proto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

110

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“…Several other studies on CaM using techniques such as far-and near-ultraviolet CD measurements, 'H NMR and fluoresence measurements have shown that Tyr-99 and Tyr-138, which are located in domains 111 and IV, are most affected by binding of the first two calciums; this is also consistent with the idea that sites I11 and IV are, in fact, the high-affinity sites for calcium (for review, see [23]). The ability of lanthanides to replace CaZ ' in a variety of proteins arises from the similar ionic size and coordinating properties of these ions [24]. For several calcium binding proteins that bind more than one calcium ion/molecule, it has been shown that terbium binds with the same site preference as calcium (e. g. troponin C [25]).…”

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confidence: 99%

“…The ability of lanthanides to replace CaZ ' in a variety of proteins arises from the similar ionic size and coordinating properties of these ions [24]. For several calcium binding proteins that bind more than one calcium ion/molecule, it has been shown that terbium binds with the same site preference as calcium (e. g. troponin C [25]).…”

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confidence: 99%

Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments

Martin

Linse

Bayley

et al. 1986

European Journal of Biochemistry

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The kinetics of cadmium and terbium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by stopped-flow fluorescence methods, using the calcium indicator Quin 2.Studies of the tryptic fragments TRIC and TR2C, comprising the N-terminal or C-terminal half of calmodulin, have clearly identified cadmium binding sites I and I1 as the low-affinity (rapidly dissociating) sites and sites I11 and IV as the high-affinity (slowly dissociating) sites. Thus the site preference of cadmium is the same as that of calcium. For terbium, however, sites I and I1 are the high-affinity sites and sites I11 and IV are the low-affinity sites. Thus, the site preference or terbium is not the same as that of calcium and cadmium.In contrast to previous studies with calcium, we observe two kinetic precesses for dissociation from sites I11 and IV for experiments with both cadmium and terbium. Possible models for the binding of metal ions are discussed.Calmodulin has four calcium binding domains, numbered I to IV starting from the N-terminus [l] in attempts to clarify the assignment of the four sites to the two classes with different affinities. These studies convincingly demonstrate that the strong binding sites for these ions are sites I and I1 and competitive titration of Tb-CaM complexes with Ca2+ appeared to show that Ca2+ was also most strongly bound at sites I and I1 [8].However, recent studies using l13Cd NMR [12], 43Ca NMR [22] and stopped-flow fluoresence measurements [14] with calmodulin and its tryptic fragments have shown that the high-affinity calcium (and cadmium) binding sites are, in fact, sites 111 and IV. Several other studies on CaM using techniques such as far-and near-ultraviolet CD measurements, 'H NMR and fluoresence measurements have shown that Tyr-99 and Tyr-138, which are located in domains 111 and IV, are most affected by binding of the first two calciums; this is also consistent with the idea that sites I11 and IV are, in fact, the high-affinity sites for calcium (for review, see [23]).The ability of lanthanides to replace CaZ ' in a variety of proteins arises from the similar ionic size and coordinating properties of these ions [24]. For several calcium binding proteins that bind more than one calcium ion/molecule, it has been shown that terbium binds with the same site preference as calcium (e. g. troponin C [25]). It is therefore surprising that these ions should show an inverted binding preference in the case of calmodulin, although, as has been noted by Wang et al. [15], such an inversion is quite common for binding of these metals to small chelators.In a recent publication [14] we showed that fluorescence stopped-flow studies of CaM and its tryptic fragments were highly diagnostic in confirming the distribution of the binding sites into two classes for the interaction of CaM with calcium. This is because tryptic cleavage results in two fragments that each contain a pair of calcium binding sites, TRIC (residues

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“…The proposed leaving site for transported protons identified in this work would have been difficult to locate without the use of the positively charged Ho 3þ to reveal a negative patch of residues important for proton unloading. Ho 3þ is a relatively small cation (ionic radii 0.96 Å) with a high charge density and well known to form complexes with the deprotonated form of carboxylates in protein structures (46,47). Our structural and biochemical data identify Ho 3þ as a novel chemical tool for structural identification of proton binding sites.…”

Section: Discussionmentioning

confidence: 87%

Structural identification of cation binding pockets in the plasma membrane proton pump

Ekberg

Pedersen²,

Sørensen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The activity of P-type plasma membrane H + -ATPases is modulated by H + and cations, with K + and Ca 2+ being of physiological relevance. Using X-ray crystallography, we have located the binding site for Rb + as a K + congener, and for Tb 3+ and Ho 3+ as Ca 2+ congeners. Rb + is found coordinated by a conserved aspartate residue in the phosphorylation domain. A single Tb 3+ ion is identified positioned in the nucleotide-binding domain in close vicinity to the bound nucleotide. Ho 3+ ions are coordinated at two distinct sites within the H + -ATPase: One site is at the interface of the nucleotide-binding and phosphorylation domains, and the other is in the transmembrane domain toward the extracellular side. The identified binding sites are suggested to represent binding pockets for regulatory cations and a H + binding site for protons leaving the pump molecule. This implicates Ho 3+ as a novel chemical tool for identification of proton binding sites.

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Lanthanides as probes for calcium in biological systems

Cited by 362 publications

References 109 publications

Crystal structure of a Trypanosoma brucei metacaspase

Crystal structure of a Trypanosoma brucei metacaspase

Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments

Structural identification of cation binding pockets in the plasma membrane proton pump

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