Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

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.

Fig 1. SAXS models of isolated AC.

(A) Adjustment of the curve calculated from the crystal structure of AC extracted from the pdb dataset 1YRU (blue curve) against experimental data (black dots). (B) Adjustment obtained by releasing the residues comprising helices F through H′ (red curve). (C) Distribution of reduced residuals corresponding to the two fits shown in panels (A) and (B), using the same color code. (D) Crystal structure (blue) and conformation with relaxed F–H′-helices (red). AC, adenylate cyclase catalytic domain; pdb, Protein Data Bank; SAXS, small-angle X-ray scattering.

Fig 2. AC dynamics are significantly altered upon CaM interaction.

HDX-MS was performed at the peptide level to improve the spatial resolution. Panel (A) displays relative fractional uptake maps of AC alone and upon CaM binding. The presence of dynamic HDX-MS indicative of structure formation is colored green, whereas non-dynamic events are colored gray. CaM binding to AC results in an overall reduction in the solvent accessibility and structure formation in several regions. (B) Multiple regions of AC are altered upon CaM binding, as determined by the uptake difference chart. Major modifications of the AC T25 region are observed within helices B, C, and F. For the T18 region, primary sites of alteration are found within the H/H″-, I-, and J/J′-helices as well as within the T18b1 and C-tail regions of the protein. No changes in accessibility were observed at the Hom- and catalytic loops in either state. Sites of ATP-binding are also given. (C) Several types of HDX-MS behavior were observed upon AC:CaM complex formation. All AC peptides selected for final HDX-MS analysis are displayed in S10 Fig. (D) Logit representation of the statistical results generated for each peptide by MEMHDX. Peptides colored red display nondynamic behavior in the AC alone state only, while those in blue give nondynamic behavior in the AC + CaM state only. Green peptides are those that have nondynamic HDX-MS behavior in both states, while those peptides colored black are dynamic in both states. The FDR value was set to 0.01 (red lines). The data used to generate the figure can be found in S1 Data. AC, adenylate cyclase catalytic domain; CaM, calmodulin; FDR, false discovery rate; MEMHDX, Mixed-Effects Model for HDX experiments; T18, C-terminal trypsin-cleavage fragment of CyaA (amino acids 225–364); T25, N-terminal trypsin-cleavage fragment of CyaA (amino acids 1–224); T18b1, first beta-sheet of the T18 fragment.

Fig 3. SAXS envelopes of CaM alone and in the presence of H-helix and P_MLCK peptides.

(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.

Fig 4. Differential HDX-MS patterns within CaM upon H-helix and AC binding.

(A) Relative fractional exchange data were calculated at each point and plotted as a function of peptide position for full-length CaM. (B) Uptake difference plot for CaM in the presence of AC. AC-induced differences in deuterium exchange are principally located in C-CaM. (C) Uptake “difference of uptake differences” plot of CaM in the presence of AC and H-helix peptide. Addition of the full-length catalytic domain results in much greater differences in the deuteration of both N- and C-CaM compared to the H-helix region alone, e.g., in peptides 84–90 and 104–113. Uptake plots of all CaM peptides selected for final HDX-MS analysis are given in S11 Fig. The data used to generate the figure can be found in S3 and S4 Data. AC, adenylate cyclase catalytic domain; C-CaM, C-terminal domain of CaM; CaM, calmodulin; HDX-MS, hydrogen/deuterium exchange mass spectrometry; N-CaM, N-terminal domain of CaM.

Fig 5. Modeling of the AC:CaM complex.

(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.

Fig 6. The structural interplay of AC and CaM complex formation.

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 S1, S3 and S4 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.