Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

O'Brien DP, Durand D, Voegele A, Hourdel V, Davi M, Chamot-Rooke J, Vachette P, Brier S, Ladant D, Chenal A, PLoS Biol 15(12):e2004486 (2017) Europe PMC

SASDCL9 – Catalytic domain (AC) of B. Pertussis Adenylate Cyclase Toxin (CyaA) in complex with calmodulin

Bifunctional hemolysin/adenylate cyclase
Calmodulin
MWI(0) 56 kDa
MWexpected 56 kDa
VPorod 78 nm3
log I(s) 3.83×10-2 3.83×10-3 3.83×10-4 3.83×10-5
Bifunctional hemolysin/adenylate cyclase Calmodulin small angle scattering data  s, nm-1
ln I(s)
Bifunctional hemolysin/adenylate cyclase Calmodulin Guinier plot ln 3.83×10-2 Rg: 2.9 nm 0 (2.9 nm)-2 s2
(sRg)2I(s)/I(0)
Bifunctional hemolysin/adenylate cyclase Calmodulin Kratky plot 1.104 0 3 sRg
p(r)
Bifunctional hemolysin/adenylate cyclase Calmodulin pair distance distribution function Rg: 3.0 nm 0 Dmax: 9.8 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Bifunctional hemolysin/adenylate cyclase Calmodulin BUNCH model

log I(s)
 s, nm-1
Bifunctional hemolysin/adenylate cyclase Calmodulin EOM/RANCH model

Synchrotron SAXS data from solutions of the catalytic domain (AC) of B. Pertussis adenylate cyclase toxin (CyaA) in complex with calmodulin in 20 mM Hepes, 150 mM NaCl, 4 mM CaCl2, pH 7.4 were collected on the SWING beam line at the SOLEIL storage ring (Saint-Aubin, France) using a CCD AVIEX detector at a sample-detector distance of 2.0 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsin θ/λ and 2θ is the scattering angle). 250 successive 1.500 second frames were collected at at 15°C using size-exclusion chromatography SAXS.

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 0.82 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 The average value is MWexperimental=56.3 kDa. AC-CAM complex: Top panel: Comparison of the experimental data (blue dots) with the calculated scattering pattern (red line) of the BUNCH model shown on the right. chi2=1.96. Each CaM domain were handled as rigid bodies while the program searches an optimal conformation of the inter-domain helix of CaM. Bottom panel: Typical ensemble of conformations describing the AC:CaM complex, obtained using the program EOM and displayed after superimposition of the AC moiety of each conformation. chi2=1.41. The corresponding scattering curve is shown in red superimposed over experimental data (blue dots).

Bifunctional hemolysin/adenylate cyclase (AC domain from CyaA)
Mol. type   Protein
Organism   Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251)
Olig. state   Monomer
Mon. MW   39.4 kDa
 
UniProt   P0DKX7 (1-364)
Sequence   FASTA
 
Calmodulin (CaM)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   16.8 kDa
 
UniProt   P62158
Sequence   FASTA