Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor

Abstract

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.

Figure 1

Structure of EF–CaM complex. (A) Ribbon diagram of EF in complex with CaM and that of LF. Catalytic core domain, helical domain, N-terminal PABD, and C-terminal PABD of EF are colored in green, yellow, blue, and purple, respectively, and CaM in red. N-terminal PABD, C-terminal PABD, and protease domain of LF are in blue, purple and green, respectively. (B) Comparison of PABDs of EF and LF. The similar secondary structures of the N-terminal α/β sandwich of PABDs of EF and LF are depicted in blue and dark blue, respectively, and those of the C-terminal five-helix domain of PABD of EF and LF are colored in purple and magenta, respectively. Five loops, L1–L5, which have significant differences between EF-PABD and LF-PABD, are colored in cyan and yellow, respectively. (C) Sequence alignment of PABD of EF and LF. Identical sequences are colored in yellow and similar sequences are in green.

Figure 2

Structures of N-CaM and its interaction with EF. (A) Structures of N-CaM (red) in EF–CaM complex at 1 μM calcium, 1 mM calcium, 10 mM calcium concentrations in comparison with the calcium-free N-CaM structure (left, PDB code: 1CFD) and the crystal structure of four calcium-loaded CaM (right, PDB code: 1CLL). Calcium ions are colored in orange. (B) The interaction between N-CaM and the helical domain of EF. The helical domains of EF and N-CaM of the EF–CaM complex at 10 mM calcium concentration are colored in yellow and red, respectively. For comparison, four calcium-loaded CaM is shown in cyan. (C) Detailed hydrogen bonding and salt bridge formed at the interface between helices I and II of N-CaM and helices L and M of EF.

Figure 3

Conformations of calcium-binding sites 1 and 2 of N-CaM. Calcium-binding sites 1 and 2 of N-CaM in the calcium-free NMR average solution structure, structures of EF–CaM in the presence of 1 μM, 1 mM, and 10 mM CaCl₂, and four calcium-loaded CaM. Oxygen, carbon, nitrogen, backbone, and calcium are in red, gray, blue, green, and orange. The simulated annealing omit map of calcium ion (cyan and pink) are contoured at the 5.0σ level.

Figure 4

The active site of EF. The comparison of active sites of EF–CaM in complex with 3′dATP (A) and the complex of EF-ACD and CaM with 3′dATP (B). The simulated annealing omit map was contoured at 3.0σ level. Oxygen, nitrogen, carbon, and metal atoms are in red, blue, black, and orange, respectively. Secondary structures of EF and ligands are in green and black, respectively. (C) The catalytic site of the simulated model based on the EF–CaM–3′dATP structure. ATP conformation and coordination of Mg²⁺ ions obtained from a representative snapshot along the MD trajectory of the EF–CaM–ATP complex in aqueous solution. Hydrogen atoms and water molecules are not shown.

Figure 5

The catalytic mechanism of EF. (A) The adenylyl cyclase activity of wild-type EF-ACD and its mutants, H351A, H351K, and H351R (0.8 nM each) in response to the activation of CaM. The assay was performed at pH 7.2 in the presence of 10 mM MgCl₂, 1 mM EDTA, 1 μM free CaCl₂, 10 μM CaM, and 10 mM ATP. (B) The adenylyl cyclase activity of wild-type EF-ACD and its mutants H351A, H351K, and H351R in response to the pH changes. Adenylyl cyclase activity was measured in the presence of 10 mM ATP, 10 μM CaM, 1.2 μM free CaCl₂, 500 μM BAPTA, and 10 mM MgCl₂. Mean±s.e. are representative of at least two experiments. (C) The active site of EF in complex with CaM and cAMP. The simulated annealing omit map was contoured at the 3.0σ level. Oxygen, nitrogen, carbon, and metal atoms are in red, blue, black, and orange, respectively. Secondary structures of EF and ligands are in green and black, respectively.

Figure 6

The comparison of class II adenylyl cyclase toxins. (A) Schematic diagram of domain organization of five adenylyl cyclase toxins. The adenylyl cyclase core domains and the conserved segments, A, B, and C are indicated. Yp ACT-A/B and Yptb ACT refer to the adenylyl cyclase toxin from Yersinia pestis and Yersinia pseudotuberculosis, respectively. Accession numbers for EF, CyaA, ExoY, Yp ACT-A, Yp ACT-B, and Yptb ACT are P40126, P15318, AAC78299, NP993432, NP993433, and YP070748, respectively. Yp ACT consists of two polypeptide chains due to the frameshift mutation. Yp ACT-B has sequence homology with bacterial DNA gyrase and oligopeptide ATP transporter. The C-terminal 145 kDa domain of CyaA can bind α_Mβ₂ integrin and has hemolytic activity (El-Azami-El-Idrissi et al, 2003). The N-terminal domains of Yptb and Yp ACT are homologous to the C locus of insecticidal toxin complex c (TccC) (Bowen et al, 1998). (B) Sequence alignment of three conserved segments of class II adenylyl cyclases. The highly conserved residues crucial for catalysis are highlighted by asterisk. EF H351 and the corresponding residues are underlined. The adenylyl cyclase activity of CyaA (C), ExoY (D) and their mutants in response to the pH changes. Adenylyl cyclase activity was measured in the presence of 10 mM ATP, 1.2 μM free CaCl₂, 500 μM BAPTA, 10 mM MgCl₂, 1 μM CaM (for CyaA) or 10 μg spleen lysate (for ExoY).

Figure 7

The proposed two-metal–ion catalytic mechanism of EF.