Interaction of Calcium with Bordetella pertussis Adenylate Cyclase Toxin

Rose, Thierry; Šebo, Peter; Bellalou, Jacques; Ladant, Daniel

doi:10.1074/jbc.270.44.26370

Cited by 154 publications

(106 citation statements)

References 38 publications

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“…CyaA is composed of a 141 kDa hemolytic C‐terminal domain and a 43 kDa N‐terminal adenylate cyclase toxin domain (CyaA‐ACD) 1, 2 When stimulated by CaM, CyaA‐ACD converts ATP to cAMP leading to a pathophysiological accumulation of intracellular cAMP 3, 4, 5. Interaction with both N‐terminal and C‐terminal domains of intact CaM contributes to a 400‐fold increase in CyaA‐ACD binding affinity as compared to the isolated N‐terminal or C‐terminal domain, but the molecular mechanism remains to be determined 6.…”

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confidence: 99%

Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin

et al. 2016

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Adenylate cyclase toxin domain (CyaA‐ACD) is a calmodulin (CaM)‐dependent adenylate cyclase involved in Bordetella pertussis pathogenesis. Calcium (Ca2+) and magnesium (Mg2+) concentrations impact CaM‐dependent CyaA‐ACD activation, but the structural mechanisms remain unclear. In this study, NMR, dynamic light scattering, and native PAGE were used to probe Mg2+‐induced transitions in CaM's conformation in the presence of CyaA‐ACD. Mg2+ binding was localized to sites I and II, while sites III and IV remained Ca2+ loaded when CaM was bound to CyaA‐ACD. 2Mg2+/2Ca2+‐loaded CaM/CyaA‐ACD was elongated, whereas mutation of site I altered global complex conformation. These data suggest that CyaA‐ACD interaction moderates CaM's Ca2+‐ and Mg2+‐binding capabilities, which may contribute to pathobiology.

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confidence: 99%

Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin

et al. 2016

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“…45 Ca (1 Ci) at various 45 Ca specific activities in Ca-EGTA buffer (10 nM to 10 mM Ca free ) was incubated with 0.825 g of CE for 1 h at 30ЊC in 1 ml of buffer (50 mM Tris⅐HCl, pH 7.4͞50 mM KCl͞5 mM MgCl 2 ͞0.01 mM EGTA) and subjected to ultrafiltration for 18 h at 4ЊC. Sample treatment and calculation of bound and free 45 Ca was performed using the method of Rose et al (24). 45 Ca Overlay.…”

Section: Purification Of Cementioning

confidence: 99%

Calexcitin: A signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability

Nelson

Cavallaro²,

Yi³

et al. 1996

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

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A previously uncharacterized 22-kDa Ca 2؉ -binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EFhands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca 2؉ , calexcitin bound GTP and possessed GTPase activity. Calexcitin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca 2؉ and the absence of endogenous Ca 2؉ ͞cal-modulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 ؎ 6 pS K ؉ channels. Calexcitin thus appears to directly regulate K ؉ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca 2؉ -activated signaling molecule that increases cellular excitability, which would in turn increase Ca 2؉ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca 2؉ .In neuronal cells, ion channel conductance is regulated by ligand binding, direct interaction with G proteins, or phosphorylation (1). K ϩ and Ca 2ϩ channels, for example, can be phosphorylated by Ca 2ϩ ͞calmodulin-dependent kinase (2, 3) and͞or protein kinase C (PKC) (4-8); however, other elements of Ca 2ϩ signaling cascades might also regulate ion channels directly. Such a protein was suggested by a previous study in which a low molecular weight protein, designated cp20, reduced two voltage-dependent K ϩ currents, i A and i Ca-Kϩ , in identified molluscan neurons (9-11). These same currents were reduced in the same neurons in molluscs (Hermissenda crassicornis) exposed to a Pavlovian conditioning paradigm (9, 12). To investigate this protein, we purified calexcitin (CE) from squid optic lobe and used microsequencing and PCR techniques to obtain a DNA probe which was used to screen a cDNA library. The cloned protein had separate regions that are homologous to Ca 2ϩ -binding proteins and to GTPbinding proteins. Functionally, this protein binds both Ca 2ϩ and GTP, is phosphorylated by PKC, and regulates K ϩ channels in human fibroblasts and membrane excitability in mammalian and molluscan neurons. CE, therefore, offers a molecular link between PKC, intraneuronal Ca 2ϩ , and persistent changes of membrane excitability that correlate with associative memory storage. METHODSPurification of CE. Squid optic lobes were homogenized in a high speed homogenizer with 1 ml of buffer (10 mm Tris⅐HCl, pH 7.4͞20 g/ml leupeptin͞50 g/ml pepstatin͞50 mM NaF͞1 mM EDTA͞1 mM EGTA) containing 1 M DTT and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication with a 20-W probe sonicator. The homogenate was centrifuged at ...

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“…One motif sequence serves as two half sites for Ca 2ϩ binding, and an array of the sequences forms the parallel ␤-roll structure, as revealed by the crystal structure of alkaline protease of Pseudomonas aeruginosa (1). Biochemical and molecular biological studies have best characterized the following RTX proteins: HlyA of Escherichia coli (4,6,31) and CyaA of Bordetella pertussis (10,11,29). CyaA is a natural fusion protein of adenylate cyclase and hemolysin and exhibits toxicity that modifies the host cellular functions by increasing the intracellular concentration of cyclic AMP (20).…”

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confidence: 99%

“…CyaA is a natural fusion protein of adenylate cyclase and hemolysin and exhibits toxicity that modifies the host cellular functions by increasing the intracellular concentration of cyclic AMP (20). The binding of Ca 2ϩ ions is essential for these proteins to acquire the toxic conformation (4,29). HlyA (K564 and K690) and CyaA (K983) are palmitoylated at the lysine residues by the acyltransferases HlyC (31) and CyaC (11), respectively.…”

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Purification and Characterization of a Hemolysin-Like Protein, Sll1951, a Nontoxic Member of the RTX Protein Family from the Cyanobacterium Synechocystis sp. Strain PCC 6803

et al. 2006

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The hemolysin-like protein (HLP) Sll1951, characterized by the GGXGXDXUX nonapeptide motif implicated in Ca 2؉ binding, was purified from the glucose-tolerant strain (GT) of Synechocystis sp. strain PCC 6803. HLP was eluted at 560 kDa after gel filtration chromatography. Atomic absorption spectroscopy indicated that the protein bound calcium. The bound Ca 2؉ was not chelated with EGTA; however, it was released after being heated at 100°C for 1 min, and it rebound to the Ca 2؉ -depleted protein at room temperature. The apparent HLP molecular mass increased to 1,000 kDa and reverted to 560 kDa during the release and rebinding of Ca 2؉ , respectively. The monomers of the respective forms appeared at 90 and 200 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HLP showed no apparent hemolytic activity against sheep erythrocytes; however, a slight hemolytic activity was detected during the conformational change caused by the rebinding of Ca 2؉ . Immunoelectron microscopy using polyclonal antibodies against the 200-kDa monomer revealed that HLP is located in the cell surface layer. The localization and Ca 2؉ -induced reversible conformational change suggest that HLP is a member of the repeat in toxin (RTX) protein family despite its latent and low toxicity. In some other cyanobacteria, RTX proteins are reported to be necessary for cell motility. However, the GT was immotile. Moreover, the motile wild-type strain did not express any HLP, suggesting that HLP is one of the factors involved in the elimination of motility in the GT. We concluded that the involvement of RTX protein in cyanobacterial cell motility is not a general feature.

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Interaction of Calcium with Bordetella pertussis Adenylate Cyclase Toxin

Cited by 154 publications

References 38 publications

Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin

Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin

Calexcitin: A signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability

Purification and Characterization of a Hemolysin-Like Protein, Sll1951, a Nontoxic Member of the RTX Protein Family from the Cyanobacterium Synechocystis sp. Strain PCC 6803

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