SASDCK9: Catalytic domain (AC) of B. Pertussis Adenylate Cyclase Toxin (CyaA)

Basic information

• Entry: Database: SASBDB / ID: SASDCK9

Sample

Catalytic domain (AC) of B. Pertussis Adenylate Cyclase Toxin (CyaA)

• Bifunctional hemolysin/adenylate cyclase (protein), AC domain from CyaA, Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251)

Function / homology

Function:

calcium- and calmodulin-responsive adenylate cyclase activity / hemolysis in another organism / adenylate cyclase / cAMP biosynthetic process / adenylate cyclase activity / channel activity / positive regulation of cytosolic calcium ion concentration / toxin activity / calmodulin binding / calcium ion binding / host cell plasma membrane / extracellular region / ATP binding / membrane

Domain/homology:

RTX, pore-forming domain / N-terminal domain in RTX protein / Haemolysin-type calcium binding-related / Haemolysin-type calcium binding protein related domain / RTX toxin determinant A / Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / Hemolysin-type calcium-binding conserved site / Hemolysin-type calcium-binding region signature. / RTX calcium-binding nonapeptide repeat / RTX calcium-binding nonapeptide repeat (4 copies) / Serralysin-like metalloprotease, C-terminal

Component:

Bifunctional hemolysin/adenylate cyclase

• Biological species: Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) (bacteria)
• Citation: Journal: PLoS Biol / Year: 2017; Title: Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.; Authors: Darragh P O'Brien / Dominique Durand / Alexis Voegele / Véronique Hourdel / Marilyne Davi / Julia Chamot-Rooke / Patrice Vachette / Sébastien Brier / Daniel Ladant / Alexandre Chenal / ; Abstract: Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes.

Contact author

• Dominique DURAND (I2BC-CNRS)

Models

• Model #1690: ; Type: mix / Radius of dummy atoms: 1.90 A / Chi-square value: 2.565; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1691: ; Type: atomic / Radius of dummy atoms: 1.90 A / Chi-square value: 1.358 / P-value: 0.000720; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Catalytic domain (AC) of B. Pertussis Adenylate Cyclase Toxin (CyaA)
• Buffer: Name: 20 mM Hepes, 150 mM NaCl, 4 mM CaCl2 / pH: 7.4

Entity #904

Name: AC domain from CyaA / Type: protein / Description: Bifunctional hemolysin/adenylate cyclase / Formula weight: 39.38 / Num. of mol.: 1
Source: Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251)
References: UniProt: P0DKX7
Sequence:

MQQSHQAGYA NAADRESGIP AAVLDGIKAV AKEKNATLMF RLVNPHSTSL IAEGVATKGL GVHAKSSDWG LQAGYIPVNP NLSKLFGRAP EVIARADNDV NSSLAHGHTA VDLTLSKERL DYLRQAGLVT GMADGVVASN HAGYEQFEFR VKETSDGRYA VQYRRKGGDD FEAVKVIGNA AGIPLTADID MFAIMPHLSN FRDSARSSVT SGDSVTDYLA RTRRAASEAT GGLDRERIDL LWKIARAGAR SAVGTEARRQ FRYDGDMNIG VITDFELEVR NALNRRAHAV GAQDVVQHGT EQNNPFPEAD EKIFVVSATG ESQMLTRGQL KEYIGQQRGE GYVFYENRAY GVAGKSLFDD GLGA

Experimental information

• Beam: Instrument name: SOLEIL SWING / City: Saint-Aubin / 国: France / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.1 Å / Dist. spec. to detc.: 1.988 mm
• Detector: Name: AVIEX / Type: CCD

Scan

Title: Catalytic domain (AC) of B. Pertussis Adenylate Cyclase Toxin (CyaA)
Measurement date: Jun 19, 2015 / Storage temperature: 10 °C / Cell temperature: 15 °C / Exposure time: 1.5 sec. / Number of frames: 250 / Unit: 1/A /

	Min	Max
Q	0.0066	0.3999

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 773 /

	Min	Max
Q	0.00663373	0.399925
P(R) point	1	773
R	0	91

Result

Type of curve: sec
Comments: The scattered intensities were displayed on an absolute scale (cm-1) using the scattering of water. Frames were examined individually and 20 identical frames were averaged and further processed. The corresponding concentration was 0.80 g/L. Three independent determinations of the molecular mass were obtained from the value of I(0)/c, where c is the protein concentration, and using the programs SAXSMow2 and ScÅtter3 available at the URLs http://saxs.ifsc.usp.br/ and https://bl1231.als.lbl.gov/scatter/, respectively. The average value is MWexperimental=40.2 ± 1.0 kDa. AC in solution: Top panel: Adjustment of the curve calculated from the crystal structure of AC extracted from the pdb dataset 1YRU (red curve) after missing loop addition using AllosModFoxs against experimental data (blue dots). chi2=2.57 Bottom panel: Adjustment obtained using AllosModFoxs by releasing residues 200-270 comprising helices F through H’ (red curve). Residues 200-270 correspond to the large yellow-light green loop seen in the model displayed on the right. chi2=1.36

	Experimental	Standard	Standard error	Porod
MW	40.2 kDa	38.6 kDa	2	40.1 kDa
Volume	-	-	-	60.2 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	0.0252	0.0002	0.0252	0.0002
Radius of gyration, Rg	2.62 nm	0.03	2.59 nm	0.03

	Min	Max	Error
D	-	9.1	0.5
Guinier point	1	66	-