Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

Abstract

Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF₃^- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.

Fig. 1. Native and activated DgcR dimers adopt distinct domain arrangements.

a DgcR domain organisation with important features and residues highlighted. The Rec and the GGDEF domains are linked by the extension (orange) of the C-terminal Rec helix. Red and yellow bars indicate the DxLT and GGDEF motif, respectively. b, c Side and top views of DgcR’ (c) and DgcR’* (d) dimers. Within one protomer, domains and important elements are highlighted by colour. The C-terminal Rec helix (α5) is coloured in gold, the wide turn in red, the N-terminal GGDEF helix (α0’) in cyan and the characteristic β-hairpin (with GGEEF sequence) in yellow. The BeF₃^- modified aspartates of the Rec domains, the bound Mg ⁺⁺ ions (green), and the 3’dGTP substrate analogues bound to the GGDEF active sites are shown in full. In both cases, the Rec domains obey twofold symmetry. The GGDEF domains are related by twofold symmetry (with the two 3’dGTP ligands opposing each other) only in DgcR’*, while in DgcR’ they are related by ~90°.

Fig. 2. Inter-domain hinge revealed by comparison of the two DgcR’ protomers.

a Difference of backbone torsion angles (Δtor) of DgcR’ and DgcR’* chains relative to chain A of DgcR’. Note that the two DgcR’ chains show a localised drastic difference in the main-chain torsion angle ψ136. b, c Detailed view of the inter-domain hinge segment of chain A (b) and B (c) of DgcR’. The wide turn with the D139xLT142 motif is highlighted in red. The 169° rotation of ψ136 drastically changes the relative angle between α5 of the Rec and α0’ of the GGDEF domain.

Fig. 3. Beryllofluoride modification induces a relative 16 ° rotation of two Rec halves.

Rigid-body 1 composed of α3, β4, α4, β5 (shown in pink and green for DgcR’ and DgcR’*, respectively) is rotated with respect to the rest (rigid-body 2, grey) as seen after super-postion of the two grey substructures. a Native and activated Rec domains with important residues shown in full viewed perpendicularly to the rotation axis of the relative rotation (orange). In DgcR’*, the beryllofluoride group forms an H-bond with T86 and an ionic interaction with K108 of DgcR’*. b Same as a, but in cartoon representation and viewed along rotation axis.

Fig. 4. Distinct packing of Rec domains upon BeF3- modification.

a Superimposed dimers with native structure in pink and activated structure in green. b, c Rec dimer of DgcR’ (b) and DgcR’* (c) with contacts between the protomers <3.2 Å in yellow. d Sequence alignment of Rec domains of DgcR and PhoB with secondary structure elements of DgcR and sequence logo derived from DgcR group 1 homologues (see Fig. 10). Asterisks indicate conserved residues involved in Rec domain activation. Below the individual sequences, lines connect residues that participate in inter-domain contacts (side-chain–side-chain interactions in black, interactions involving the main-chain in blue). Red dots indicate D104 and R118 of the conserved intermolecular salt-bridge.

Fig. 5. Coiled-coil linker adopts two alternative registers depending on activation state.

a Side view of parallel coiled-coil linker of DgcR’ (left) and DgcR’* (right) with residues that form contacts with their symmetry equivalents in CPK representation. Only residues from the left helix are labelled. Bold labels indicate residues that are involved in both registers (persistent contacts). b Two alternative heptad repeat patterns (a d a d and a e a e), which are adopted by DgcR’ and DgcR’*, respectively. Positions a are used in both registers (persistent contacts), whereas positions d or e are used only in the native or activated conformation (conditional contacts). Note, that the a e a e pattern can formally also be described by a d’ a’ d’ a’ pattern as indicated in the figure and used in (Gourinchas et al.). c Helical net representation of coiled-coil interactions in DgcR’ (left) and DgcR’* (right). Of the front helix, only interacting residues are shown (highlighted by colour). Interacting residues pairs are outlined in blue. d, e Top view of coiled-coil after superposition of left helix. DgcR’ and DgcR’* are shown in pink and green, respectively. For clarity, residues forming persistent and conditional contacts are shown in the separate panels d and e, respectively.

Fig. 6. The activated Rec dimer allows formation of a catalytically competent GGDEF dimer.

a Changes in main-chain dihedral angles (Δtor) applied manually to DgcR’* to move the two GGDEF domains into catalytically competent arrangement. b Model of competent DgcR (two orthogonal views) generated as described in a. c Detailed view of the competent juxtaposition of the two GGDEF bound GTP substrate molecules. The carbon atoms of the two GTP molecules are coloured in orange and grey, respectively. The O3’ hydroxyl of each ligand is poised for nucleophilic attack on the α-phosphorous (PA) of the other ligand being roughly inline with the scissile PA–O3A bond.

Fig. 7. Structural transitions in DgcR upon activation.

Rec pseudo-phosphorylation induces steps 1 to 3, which are followed by GGDEF hinge motions of steps 4 and 5 to attain the catalytically competent state. See also Supplementary Movies 1 and 2. The structures are represented as in Fig. 1b, c, but with the residues of the conditional coiled-coil contacts shown as CPK models (residues in d and e position are shown is pink and green). The beryllofluoride moieties of the dimer are highlighted by magenta circles. a DgcR’, symmetrized version with both GGDEF domains in B-chain orientation (cf. with Fig. 1b). b As in a, but with beryllofluoride-induced tertiary change applied to Rec rigid_body 1 (see Fig. 3). c As in b, but with quaternary change applied to Rec domains. Note the clash between the C-terminal ends of the coiled-coil (red circle). d As in c, but with Rec dimer as found in symmetrized version of DgcR’*. e Symmetrized version of DgcR’* (cf. with Fig. 1c). f Model of catalytically competent DgcR as in Fig. 6b.

Fig. 8. Dimeric c-di-GMP cross-links the GGDEF domain of the dimer.

a Side and top views of the DgcR/c-di-GMP complex (DgcR_inh). Representation as in Fig. 1b. b, c Detailed comparison of the c-di-GMP binding mode in DgcR (b) and PleD (2V0N) (c). Protomers are distinguished by colour hue. H-bonds are shown as green broken lines.

Fig. 9. Enzyme kinetics of DgcR.

a–c Enzymatic progress curves of wild-type DgcR and inhibition relieved mutant DgcR’ in the native and in the activated (indicated by asterisk) state. Experiments were performed with 5 μM enzyme and 500 μM GTP substrate concentrations. Symbols denote experimental values, continuous lines represent fit of the kinetic model shown in panel d to the data with parameters listed in Supplementary Table 2. a Progress curve of c-di-GMP production catalysed by DgcR* as measured by conventional IEC. b Progress curves as measured by oIEC catalysed with the indicated DgcR variants/states. c Zoom- in of b. d Kinetic model of diguanylate cyclase activity controlled by non-competitive product binding. Substrate (S) binding to the dimeric enzyme (EE) is modelled with the equilibrium dissociation constant K_d and assumed to be unaffected by the presence of S in the second binding site or of product (P) in the allosteric site. Product binding is modelled kinetically with rate constants k_on and k_off. Note that the model considers simply one instead of four product binding sites on the enzyme. Only the Michaelis–Menten complex with two bound substrate molecules and no bound product (SEES) is competent to catalyse the S + S → P condensation reaction (with turn-over number k_cat). Source data are provided as a Source Data file.

Fig. 10. The linker helices of Rec-GGDEF proteins show heptad repeat patterns and discretized lengths.

a Sequence logos of inter-domain linkers grouped according to length. Logos 1 to 6 correspond to peaks 1 to 6 in the histogram of panel b. DgcR belongs to group 1. The grey rectangles symbolise the predicted α5 helices, black bars indicate recurring hydrophobic positions spaced with a distance of 7. Data were compiled from 1991 Rec-GGDEF sequences, see Methods. b Histogram of inter-domain distances as measured from Rec KP motif to GGDEF DxLT motif. c Overall logo of C-terminal part of all sequences shown in panel a. Positions engaged in parallel coiled-coil interactions in DgcR’ and DgcR’*, are indicated in pink and green, respectively (see also Fig. 5b).