Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins

Abstract

Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca(2+) concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+) concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca(2+) concentrations. Despite the similarity in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N- and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.

Fig. 1. The effect of calcium ions and CaM on the adenylyl cyclase activities of EF and CyaA-N. Adenylyl cyclase assays were performed in the presence of 1 nM EF (A) and 0.7 nM CyaA-N (B) under 10 µM CaM (filled circles), 0.1 µM CaM (open circles) and 1 nM CaM (filled triangles, CyaA-N only) at increasing [Ca²⁺]. They were also performed at 0.1 µM Ca²⁺ (filled circles), 0.3 µM Ca²⁺ (open circles) and 1.0 µM Ca²⁺ (filled triangles) at increasing [CaM] in the presence of 1 nM EF (C), and 0.7 nM CyaA-N (D). Maximal adenylyl cyclase activities (100%) for EF in the calcium titration are 1140 s^–1 (10 µM CaM) and 228 s^–1 (0.1 µM CaM) (A) and those for CyaA-N are 1465 s^–1 (10 µM CaM), 713 s^–1 (0.1 µM CaM) and 556 s^–1 (1 nM CaM) (B). Means ± SE are representative of at least two experiments.

Fig. 2. The binding of 2′d3′ANT-ATP to EF. (A) Equilibrium titration of 2′d3′ANT-ATP–CaM with EF and EF-F586A. (B) Calcium titration of fluorescence enhancement by EF–CaM. 2′d3′ANT-ATP was added to a final concentration of 0.5 µM and the indicated free calcium concentrations were achieved by buffering with 10 mM EGTA. λexc = 320 nm and the optimal fluorescence emission of EF–CaM–2′d3′ANT-ATP (412 nm) was normalized to give the fold of enhancement. (C) Secondary structure of EF–CaM–2′d3′ANT-ATP in comparison with EF alone. (D) The active site of EF in the presence and absence of CaM and 2′d3′ANT-ATP.

Fig. 3. The effect of calcium ions on the interaction of EF and CyaA-N with immobilized CaM by SPR sensorgram analysis. Sensorgrams were recorded in the presence of 1.4 µM wild-type EF (A), 1.3 µM EF-K525A (B), 0.37 µM CyaA-N (D) and 8.0 µM EF-triple mutant (C) at the indicated free calcium concentrations. (E) Kinetic analysis of SPR sensorgrams based on a ‘two-state conformational change’ model. At the lowest calcium concentration, signal from the bound EF could be reduced to the base line with the wash using the same calcium concentration. However, a fraction of the signal could not be reduced at the higher calcium concentrations until the buffer with the lowest calcium concentration was used in the wash step. The signal from the bound CyaA-N could not be reduced to the base line without a prolonged wash.

Fig. 4. The activation of EF and CyaA-N by wild-type CaM and two series of CaM mutants. EF (1 nM) and CyaA-N (0.7 nM) were used for an adenylyl cyclase activity assay in the presence of 0.1 µM free Ca²⁺ with CaM mutants CaM 41/75 and 85/112. Each mutant has two cysteine mutations to lock either the N- or the C-terminal domain of CaM in the closed conformation (A and B). The same concentrations of EF and CyaA-N were used for an adenylyl cyclase activity assay in the presence of 1 µM free Ca²⁺ with CaM mutants B1Q, B2Q, B3Q and B4Q. Each mutant has a mutation inactivating one of four calcium-binding sites (C and D). Means ± SE are representative of at least two experiments.

Fig. 5. Effect of calcium and magnesium ions on adenylyl cyclase activity of EF and CyaA-N (A and B) and an EF mutant, EF-H577N (C and D). Adenylyl cyclase activity assays were performed with 10 µM CaM at 0.1 µM Ca²⁺ (filled circles), 0.3 µM Ca²⁺ (open circles) and 1.0 µM Ca²⁺ (filled triangles) in the presence of 1 nM EF (A), and 0.7 nM CyaA-N (B). To analyze the mutant form of EF, adenylyl cyclase activities were measured with 10 µM CaM and 1 nM EF or 66 nM EF-H577N with either 0.3 µM Ca²⁺ (C) or 10 mM Mg²⁺ (D). Both were buffered by 10 mM EGTA. Maximal activities for EF in the magnesium titration are 1616 s^–1 (0.1 µM Ca²⁺), 1074 s^–1 (0.3 µM Ca²⁺), and 1009 s^–1 (1.0 µM Ca²⁺) (A) and those for CyaA-N are 2106 s^–1 (0.1 µM Ca²⁺), 1674 s^–1 (0.3 µM Ca²⁺) and 1385 s^–1 (1.0 µM) (B). Maximal activities for EF and EF-H577N were 1208 s^–1 and 4 s^–1 (C) and those for EF and EF-H577N were 2726 s^–1 and 6 s^–1 (D), respectively. Means ± SE are representative of at least two experiments.

Fig. 6. The activation of EF and CyaA-N by wild-type CaM, N- and C-terminal domain of CaM (N-CaM and C-CaM). (A) Coomassie Blue staining of purified EF, EF mutants, CyaA-N, CaM and CaM mutants (2 µg each) on SDS–PAGE. (B) EF (1 nM), EF-ΔH (80 nM) and CyaA-N (0.7 nM) were assayed for adenylyl cyclase activity at 1.0 µM free Ca²⁺ and 10 µM CaM in the absence or the presence of full-length CaM, N-CaM, C-CaM or both N-CaM and C-CaM (N+C CaM). (C and E) Activation of CyaA-N and EF by wild-type and mutant CaM. Adenylyl cyclase toxin (0.7 nM CyaA-N or 1 nM EF) and 1.0 µM free Ca²⁺ were used in an adenylyl cyclase activity assay in the presence of wild-type CaM (filled circles), C-CaM (open circles) and N-CaM (filled triangles). (D and F) Calcium titration assay in CaM activation of CyaA-N and EF. Adenylyl cyclase activity of CyaA-N (0.7 nM) was measured with 10 µM wild-type CaM (filled circles), C-CaM (open circles) and N-CaM (filled triangles) and that of EF (1 nM) was examined with 1 µM wild-type CaM (filled circles) and 10 µM N-CaM (filled triangles). Maximal activity for wild-type CaM, N-CaM, and C-CaM is 1465, 230 and 1219 s^–1, respectively. Means ± SE are representative of at least two experiments.

Fig. 7. Calcium–CaM activation of EF and EF-triple mutant. The locations of E609, R613 and E616 and their interacting residues, R718 and N633, are shown in the ribbon representation of EF structure in the absence (A) and presence (B) of CaM. C_A–C_B domain, switch C, helical domain and CaM are colored in green, purple, yellow and red, respectively. Ca²⁺ titration assay in the presence of 10 µM CaM (C) and CaM titration assay at 0.03, 1 and 10 µM calcium (D) were performed with 1 nM EF and 0.5 nM of EF-triple mutant.