PMC

Use of EF conformational transition to search for allosteric inhibitors.(A) Workflow of the inhibitor identification; (B) Rational structural basis for an inhibition established by mean of an allosteric interaction. EF and CaM are drawn in cartoon and the TUA compound in licorice. CaM is colored in lime green, and the SABC pocket in yellow.

ATP cyclization mechanism. The arrangement of the active site suggested by crystallographic structures with (A) the basic cyclization reaction; (B) substrate and one metal ion (based on PDB structure 1K90); (C) products and either one and two metal ions (based on PDB 1SK6); and (D) the substrate and two metal ions (based on PDB 1/XFV). For clarity, all ions are represented as Mg²⁺, the actual catalytic ion, and the substrate analogs are shown as restored to ATP.

Examples of potent inhibitors of EF toxin component. Inhibitors in panel A to D were identified by targeting EF active site. (A) ethyl 5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, []; (B) (9-[2-[[bis[(pivaloyloxy)methoxy]phosphinyl]methoxy]ethyl]adenine; or bis-POM-PMEA, []; (C) 3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid, [,]; (D) MANT-CTP and propyl-ANT-ATP, []; (E) 4-[4-(4-dichloro-phenyl)-thiazolylamino]-benzenesulfonamide (10506-2A) was first identified in a cell based assays and then selected for its ability to block EF–CaM binding. 4-[[4-(4-nitrophenyl)-2-thiazolyl] amino]-benzenesulfonamide (nitro10506-2A) was identify as a non-toxic derivative of the former []. They appeared to bind the helical domain of EF, thus being allosteric as the compounds of the TUA family, which will be described below (Figure 5, and ref. []).

X-ray crystallographic structure of the EF-CaM complex []. (A) EF and CaM are displayed in cartoon representations. Calmodulin (CaM) is in green lime and loaded with two calcium ions (spheres in orange). The helical domain Hel of EF is in wheat, the CA and CB domains that form the catalytic core of EF are in marine and purple respectively. The three switches SA, SB and SC are colored in cyan, magenta and red. The catalytic residues and the ligand 3'-deoxy ATP are drawn in sticks and the Yb³⁺ ion is drawn as a magenta sphere; (B) Cartoon and surface representations of CaM structure are displayed from different views. On the left, the eight helices of CaM are labeled: A, B, and C in N-CaM; D and E in the interlinker; and F, G, and H in C-CaM. Calcium-binding loops S1 and S2 in N-CaM, S3 and S4 in C-CaM are indicated. Two Ca²⁺ ions are bound to S3 and S4 (spheres in orange). On the right, CaM hydrophobic patches as defined by Yang et al. [] are colored in yellow and the other hydrophobic residues are colored in forest green.

Calcium effect on the EF-CaM complex.Energetical influences and dynamical correlations computed in the EF-CaM complex. (A) the energetical influences between the different domains of EF (in blue) and CaM (in green) are drawn as arrows whose color and thickness indicate the intensities of the influences, as seen by MMPBSA energy dependency maps []; (B) the residues of the EF-CaM complex are represented by their C-α atoms and colored in cyan when their dynamical correlations are low (<0.5), in green when their correlations are mean (0.5–0.6) and in magenta when their correlations are high (>0.6). In the 2 Ca²⁺ bound complex (in the middle), the energetical influences are located in the vicinity of the EF/CaM interface and the most residue are correlated. Removal of calcium (on the left) reduces the energetical influences and a region of uncorrelated residues appears at the interface between the two proteins. Upon calcium addition (on the right), new energetical influences are observed even at long distances, and three sets of highly correlated residues were observed; (C) Interface between the Hel domain of EF and N-CaM in the EF-CaM complex. The conformations taken after 8 ns of MD simulations starting from 1K93 (on the left, chains C and F) and 1XFX (on the right, chains A and O) are represented in cartoon. EF Hel domain is colored in wheat with the inter L–M loop highlighted in yellow. N-CaM is colored in green lime. Residues involved in the EF/CaM interaction and residues forming CaM calcium binding sites S1 and S2 are drawn in sticks. Calcium ions are displayed as orange spheres. The inserts represent the electron density maps of the starting crystallographic structures, contoured at 1.10 s; (D) Principles of locally enhanced sampling (LES) methods; a snapshot from the LES simulations (protein in cartoons, ions in green spheres): to enhance the probability of calcium dissociation events, one Ca²⁺ ion is replicated in multiple copies; (E) Principles of free energy perturbation (FEP). The thermodynamic cycle used in the FEP method to compare calcium dissociation free energies between two protein conformational states (1k93-4Ca and 1xfx-4Ca complexes).