Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Abstract

Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 microM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.

Fig. 1.

Scheme of the virtual screening approach used to prevent the CaM activation of EF. The pocket to which the ligand was docked is shown in yellow (483 and in the inactive and active forms, respectively), EF and CaM are drawn in cartoon format. Switches A, B, and C in EF are highlighted in blue, orange, and magenta, respectively.

Fig. 2.

Projections onto the first normal modes of the PCA analysis of the conformational transition path. Projections of successive path conformations from the EF open to the closed conformation are colored from light gray to black. The conformers 8, 28, and 47 are labeled in red, as is the starting point (1). Projections of the EF conformations sampled during MD simulations recorded on the EF-CaM complex in the presence of 0, 2, and 4 Ca²⁺ and on free EF are shown in cyan, green, orange, and purple, respectively. The mean MD conformation projection (Filled Square) is shown, surrounded by an ellipsoid illustrating the scattering of the projections along the trajectory. Considering the number of built intermediate points or the number of energy evaluations as an equivalent of a MD step, the total sampling used to build the path correspond to 300 ns or 8 μs of MD, respectively.

Fig. 3.

SABC deformation along the EF transition path. The SABC pocket was detected at the interface of switches A (in green to cyan), B (in blue to indigo), and C (in magenta to red): (A) top view of the surface of SABC in conf 1 (cartoon representation); (B–E) licorice representation of SABC in conf 1 (B), 8 (C), 28 (D), and 47 (E) with the surface of the pocket displayed in transparent gray. Pocket residues are colored according to their numbers in the protein sequence, from green through blue to red. The rmsd from the initial conformation of the residues defining the SABC pocket was 0, 3.1, 6.4, and 7.3 Å, for conf 1, 8, 28, and 47, respectively.

Fig. 4.

Inhibition of EF activity by various thiophen ureidoacid (TUA) derivatives. EF (0.5 nM) was preincubated for 20 min with the indicated concentrations of TUA compounds, then 2 μM CaM was added and enzyme activity was measured 10 minutes later, as described in SI Materials. Data in the second panel (EF-CaM) correspond to enzyme activities measured when EF was preincubated with CaM prior to the addition of 100 μM TUA derivatives. Data in the third panel (CyaA) correspond to CyaA (0.2 nM) preincubated 20 min with 100 μM of TUA compounds before addition of 2 μM CaM and measure of the activity 10 min later.