AC binding to the CaM/tethered lipid bilayer structures.

<p>The different surface constructions (in a, amine-coated surface alone; in b, CaM-coated surface at a density of 45±5 ng/cm², in c and d, CaM-coated surface (at 45±5 ng/cm²) overlaid with a continuous tethered bilayer membrane, <i>t</i>BLM) were incubated with AC (48 nM or 240 nM) in HBS buffer for 30 min, and extensively washed first with HBS buffer, then with 0.1% Triton X-100 and finally with HBS buffer again. In d, the <i>t</i>BLM was disrupted by washing with 0.1% Triton X-100 before incubation with AC. AC binding to different surface constructions was monitored by measuring its enzymatic activity (expressed as initial rate of cAMP formation) with a colorimetric assay.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019101#pone.0019101-Laine1" target="_blank">[19]</a> The results shown are the mean from 6 independent experiments.</p

Evaluation of the lipid assembly fluidity.

<p>(A) Fluorescence photobleaching images as a function of time for lipid assemblies formed onto cysteamine-coated surface with different CaM coverages (45, 65 and 110 ng/cm²). (B) Fluorescence recovery curves after photobleaching of tethered lipid assemblies built on the surface with different CaM surface coverage: 45, 65 and 110 ng/cm².</p

CaM immobilization on cysteamine gold surfaces.

<p>Amount of immobilized CaM per surface (ng/cm²) as a function of the concentration of the CaM solution (µg/mL) injected over the amine-coated gold surface. The protein surface coverage was calculated from SPR measurements; the averages and standard deviations were calculated from 8 independent measurements.</p

Tethered membrane fluidity.

<p>The lipid diffusion coefficients and the mobile fractions of the biomimetic constructions are given as a function of the initial CaM coverage. They are calculated from six independent measurements.</p

Scheme of the tethered bilayer formed over CaM molecules immobilized on top of an amine-coated surface.

<p>Scheme of the tethered bilayer formed over CaM molecules immobilized on top of an amine-coated surface.</p

SPR quantification of AC binding to the different type of surfaces.

<p>AC binding to the different type of surfaces: cysteamine monolayer (red curve); cysteamine monolayer partially derivatized with CaM at a surface coverage of 45±5 ng/cm² (blue curve); and tethered bilayer assembled over the CaM coated surface (at 45±5 ng/cm²; green curve). The amounts of AC bound to the different surfaces (in ng/cm²) as a function of the AC concentrations injected onto the surfaces, were determined by SPR spectroscopy. The average and standard deviation values were calculated from three independent measurements.</p

AC activation by the immobilized CaM.

<p>(A) Initial rates of AC activity (v_i in nmoles of cAMP/min) as a function of AC concentration (nM) in the solution deposited (or injected) on the indicated surfaces: cysteamine coated surface (red curve) and cysteamine-coated surface bearing 45±5 ng/cm² of immobilized CaM (blue curve). The data and standard deviations were from at least six measurements with independent surfaces. (B) AC binding to the immobilized CaM was not impaired by the membrane solubilization procedure. Initial rates of AC activity (v_i in nmoles of cAMP/min) as a function of AC concentration in the solution (nM) deposited (or injected) onto the calmodulin coated amino-surfaces (45±5 ng CaM/cm²), were quantified either after washing of the surface with 5 mL of HBS buffer (white bars) or after washing of the surface with 2 mL of 0.1% Triton X-100 in HBS buffer followed by 5 mL of HBS buffer (blue bars). The enzymatic reaction medium (600 µL) was deposited onto the surface after removal of the HBS buffer and kinetics of Pi production were determined as mentioned above. The average and the standard deviations are from three measurements on independent surfaces.</p

The structural interplay of AC and CaM complex formation.

<p>Specific regions within the AC T18 domain serve as a MoRF for CaM recognition, binding, and activation of AC itself. (A) In the absence of CaM, the F-, G-, H-, and H′-helices and Hom-loop are found as an extended disordered coil, acting as a bait for CaM capture. (B) An interplay between protein structural disorder and order is requisite for activation by CaM of AC catalytic function. Upon CaM binding, the H- and H′-helices undergo extensive structure formation, resulting in a conformation that is appropriate for catalytic activation. Helices F and G and the Hom-loop remain unstructured throughout. Some regions become “blocked” and resistant to deuteration (highlighted in purple). (C) The effect of CaM binding on AC. (D) The effect of AC on CaM. CaM binding to AC results in widespread perturbations, primarily within the T18 region of the protein, while AC primarily binds to C-CaM, with only a transient interaction in N-CaM. In addition to those effects occurring in the H/H′ region, long-range allosteric remodeling is observed at the site of catalysis, which becomes more stable and rigid. Meanwhile, the catalytic loop does not undergo any dramatic structural rearrangement, remaining unstructured and exposed regardless of CaM availability. This is suited to a maximal turnover of ATP substrate and thus maximal toxicity in the form of cAMP production. The data used to generate the figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004486#pbio.2004486.s017" target="_blank">S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004486#pbio.2004486.s019" target="_blank">S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004486#pbio.2004486.s020" target="_blank">S4</a> Data. AC, adenylate cyclase catalytic domain; C-CaM, C-terminal domain of CaM; CaM, calmodulin; MoRF, molecular recognition feature; N-CaM, N-terminal domain of CaM; T18, C-terminal trypsin-cleavage fragment of CyaA (amino acids 225–364); T18b1, first beta-sheet of the T18 fragment; T18b2, second beta-sheet of the T18 fragment.</p

Modeling of the AC:CaM complex.

<p>(A) 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 (green) (see main text for details). (B) Corresponding fit (red curve) to experimental data (black dots). AC, adenylate cyclase catalytic domain; CaM, calmodulin; EOM, Ensemble Optimization Method.</p

SAXS envelopes of CaM alone and in the presence of H-helix and P_MLCK peptides.

<p>(A, B, and C [top panels]) DAMMIN models of CaM alone, CaM:H-helix, and CaM:P_MLCK complexes, respectively. (A, B, and C [bottom panels]) Corresponding fits (color curves) to experimental data (black dots). (D) Green curve: comparison of experimental data (black dots) to the scattering pattern of the crystal structure of CaM (pdb 1CLL) calculated using Crysol. Red curve: fit obtained using the program EOM and corresponding to the ensemble of four conformations shown in the inset after superimposition of the N-terminal domain of each conformation. (E) Comparison of the three distance distribution functions obtained using the program GNOM for CaM alone (grey), CaM:H-helix (red), and CaM-P_MLCK (cyan) complexes. (F) Comparison of experimental data (black dots) to the scattering pattern of the crystal structure of CaM: P_MLCK (pdb 2K0F shown in the inset) calculated using Crysol (blue line). The P_MLCK peptide is shown in purple. CaM, calmodulin; EOM, Ensemble Optimization Method; pdb, Protein Data Bank; P_MLCK, myosin light-chain kinase peptide; SAXS, small-angle X-ray scattering.</p