Diadenylate cyclases and c-di-AMP synthesis

DACs were detected in a wide range of different bacterial species. So far five different classes of DACs have been identified (DisA, CdaA, CdaS, CdaM, and CdaZ) many in Gram-positive bacteria belonging to the phyla of Firmicutes and Actinobacteria but also in Gram-negative bacteria and archaea (Romling 2008; Corrigan Rebecca M and Gründling 2013; Blötz et al.

2017; Commichau et al. 2019). These different classes of DACs share the highly conserved

diadenylate cyclase domain (DAC domain) accompanied by different types of regulatory do-mains (Fig. 1) (Witte et al. 2008; Corrigan Rebecca M and Gründling 2013; Commichau et al.

2015b; Rosenberg et al. 2015).

In contrast to many pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Staphylococcus pneumonia) that possess only a sole class of DACs, some bacteria are equipped with for example three different classes like bacteria of the order Bacillales. Bacillus subtilis for instance is known to carry the DAC prototype DisA, a DNA-damage sensing protein (Oppenheimer-Shaanan et al. 2011). In response to DNA lesions the synthesis of c-di-AMP is reduced leading to a delay in sporulation while an elevated intracellular c-di-AMP level stim-ulates spore formation (Bejerano-Sagie et al. 2006; Oppenheimer-Shaanan et al. 2011). The second DAC type, c-di-AMP synthase S (CdaS), was reported to be exclusively needed for the successful germation of spores in the order Bacillales, yet its function and regulation is still not well understood (Corrigan Rebecca M and Gründling 2013; Mehne et al. 2013; Mehne et al.

2014). The third DAC domain protein in B. subtilis is the most abundant and conserved class of DACs represented by CdaA (Romling 2008; Luo Y and Helmann 2012). Interestingly the deletion of all three DACs is lethal for the survival of B. subtilis emphasizing the essentiality of c-di-AMP (Luo Y and Helmann 2012; Bai et al. 2013; Mehne et al. 2013; Witte et al. 2013).

Thus far, DisA is the only DACs which was crystallized with its product c-di-AMP enabling a better understanding of the DAC reaction mechanism. DisA is a homo octamer composed of two “head-to-head” tetrameric DAC domain rings and an N-terminal part described as the DNA binding domain (HhH domain). The catalytic site is positioned between the interface of the tetrameric rings, where two DAC domain monomers are facing each other in order to form one reaction center (Fig. 2 A & B) (Witte et al. 2008). Each DAC dimer was described to form one c-di-AMP molecule and two pyrophosphates out of two ATP molecules in a metal-ion dependent manner (Mg²⁺or Mn²⁺).

Figure 1: Diadenylate cyclase domain (DAC domain) organization of the different classes. The different do-mains are characterized by a colour code. The highly conserved DAC do-main is represented in blue. HhH, helix-hinge-helix domain; TM, transmem-brane domain; cc, coiled-coil domain;

H1 and H2, inhibitory helix 1 and 2;

PYK, pyruvate kinase-like domain (modified from Commichau et al.

2019).

By sequence alignment three highly conserved amino acid motifs were identified in the nucle-otide binding pocket. Structural and biochemical analyses demonstrate the involvement of these amino acids in nucleotide binding and catalysis (for DisA: D⁷⁵GA, T¹⁰⁷RHR, S¹²⁷) (Witte et al. 2008). Crystallization of DisA in complex with an ATP analogue enabled the characteri-zation of its pre-reaction state and the description of a detailed reaction mechanism (Müller et al. 2015).

Figure 2: Crystal structure of DisA and active site. (A) Overall octameric DisA (PDB code: 3C21) structure with a central DAC domain (molecule A light and molecule B dark blue) and the C-terminal DNA binding HhH domain (cyan) depicted as a cartoon model. Both functional domains of the protein are linked by a helical spine linker (dark and light grey). (B) DisA cartoon model of the two protomers forming the central, functional DAC unit with a bound c-di-AMP. The colour code as described in A (PDB code: 4YVZ). (C) Pre-reaction state with bound ATP analogue 3’-deoxyATP and a Mn²⁺ ion. The ATP is displayed in ball and stick mode (carbon in yellow, phosphates in orange, oxygens in red, and nitrogen in blue). Shown are amino acids that are involved in metal ion and phosphate coordination. The two DAC domains that are facing each other are coloured in light and dark blue. (D) Post-catalytic state with c-di-AMP bound. C-di-AMP is depicted in ball and sticks (carbon in light blue and dark blue, phosphates in orange, oxygens in red, and nitrogen in blue). All amino acids involved in purine base coordination are shown as sticks.

The N1 nitrogen of the nucleotide adenine is forming a hydrogen bond with the amide of the leucine main chain at position 94 while the N6 amine is hydrogen bonded by the leucine main chain carbonyl and the threonine 111 side chain (Fig. 2C). The three phosphates of the ATP analogue (3’deoxyATP) are bent around a catalytic metal ion. While the b- and g-phosphate are additionally coordinated by the Arg¹⁰⁸, His¹⁰⁹, Ser¹²⁷ and Arg¹³⁰ through hydrogen bonds, the a-phosphate interacts with Thr¹⁰⁷ and Asp⁷⁵ of the opposite monomer (Fig. 2D). An inter-action of the g-phosphate with the amino acids Ser¹²⁷, Arg¹²⁸ and Arg¹³⁰ result in its polarization

which is described as the preparation of the first reaction step (Müller et al. 2015). The reaction mechanism was reported as a two-step synthesis with two transition state complexes (Fig. 3).

The polarized g-phosphate facilitates the nucleophilic attack of the ribose 3’OH on the a-phos-phate on the neighboring ATP molecule resulting in the release of the first pyrophosa-phos-phate and the formation of a linearized intermediate (pppApA). The second step is described as an addi-tional nucleophilic attack of the second ribose 3’OH and a-phosphate which is facilitated by a complex formation of the deprotonated pppApA with the catalytic metal ion (Mg²⁺ or Mn²⁺).

This process results in the cyclization of two ATP molecules and therefore the formation of c-di-AMP (Manikandan et al. 2014). A similar mechanism was also reported for the enzyme cyclic GMP-AMP synthase (cGAS) (Ablasser et al. 2013; Kranzusch 2019).

In comparison to ATP, c-di-AMP is less coordinated. In the pre-catalytic state, the phosphates mainly contribute to the coordination of the nucleotide while the post-catalytic state shows less interaction points in order to facilitate product release (Fig. 3) (Müller et al. 2015). The guani-dine group of arginine 108 which is positioned in one of the conserved amino acid patches binds the ribose via stacking. In addition, the ribose 3’ hydroxyl is forming a hydrogen bond with the amide nitrogen of glycine 76 located in the first conserved amino acid motif (DGA) and the aspartic acid 75 is positioned in the vicinity of the phosphate moiety. The adenine moiety of c-di-AMP is coordinated as described for the ATP analogue binding (Witte et al.

2008; Müller et al. 2015).

Figure 3: The two-step mechanism of c-di-AMP synthesis. c-di-AMP is synthesized out of two ATP molecules in a metal ion-dependent manner. The first synthesis step describes the nucleophilic attack by the 3’OH group of one ATP on the a-phosphate of the opposite ATP molecule which results in the intermediate I pppApA and the release of PPi. This follows a second synthesis steps, which involves the intermediate II (pppApA in complex with Mn²⁺). A similar nucleophilic attack results in formation of c-di-AMP and the release of PPi. Important residues are depicted in stick mode (carbon in light blue, oxygens in red, and nitrogen in blue) generated with pymol (modified from Opoku-Temeng C. et al. 2016)(Manikandan et al. 2014, Schrödinger L.L.C. 2010).