Structural basis of adenylyl cyclase 9 activation

Abstract

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

Fig. 1. Identification, biochemical properties and structure of DARPin C4 as a partial activator of AC9.

a–b AC9 activity screens identify the DARPin C4 as an AC9 activator; the experiment in (b) was performed in the presence of GTPγS-bound Gαs (data are shown as mean values, n = 2). Here and in all other panels n refers to independent experiments. Source data are provided as a Source Data file. c, Activity assays of purified, detergent-solubilized ACs (as in (a, b)) demonstrate selectivity of DARPin C4 for AC9 (data are shown as mean ± SEM, n = 3 for all datasets; n = 4 for AC9 and AC9₁₂₅₀). The values were compared using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test; for AC9 vs AC9 + C4, P = 0.0002 (***), for AC91250 vs AC9₁₂₅₀ + C4, P < 0.0001 (***). P values for all comparisons are available in the Source Data file. d Dose–response activity curves of the DARPin C4 show that it has a high apparent affinity for AC9 (left) and for AC9₁₂₅₀ (right); unlike Gαs, DARPin C4 acts as a partial activator of AC9 regardless of the presence of the C2b domain (data are shown as mean ± SEM, n = 3; for AC9₁₂₅₀ + C4, n = 4). e AC activity assays performed using membrane preparations of Sf9 cells expressing different ACs confirm that activation by DARPin C4 is selective for AC9 (data are shown as mean ± SEM, n = 4). The values were compared using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons. For AC9 vehicle vs C4, P = 0.0091 (**); for vehicle vs D4, P = 0.9986; Gαs data were excluded in analysis). f Cryo-EM map of the DARPin C4-bound AC9 (white map corresponds to AC9, red—DARPin C4. g Model of the AC9–C4 complex; key elements of the structure, including the transmembrane domain bundle (TM), the helical domain (HD), the catalytic domain (CAT) and the DARPin C4 (C4, red) are indicated.

Fig. 2. DARPin C4-binding site within the catalytic domain of AC9.

a–c Views of the DARPin-C4- (a) and Gαs-bound AC9 (b). c The binding sites of the two interaction partners of AC9 overlap. The symbols in (c) indicate the points of view in (d) and (e). d–e The views of the activator binding sites in the DARPin C4- (orange) or Gαs-bound (blue) AC9 complexes. The side-chain of all residues within 4 Å of the interaction partner are shown as sticks. f–g The views of the AC9–activator interface, revealing the absence (f, indicated by an asterisk) and the presence (g) of interaction between AC9 and DARPin C4 and Gαs, respectively, involving the C1a domain loop region (residues A381 and F382 in proximity with G protein are indicated).

Fig. 3. Experimentally observed density in the nucleotide-binding site of AC9.

a The portion of the density corresponding to the bound nucleotide (MANT-GTP) in the map AC9–C4 is shown as gray surface; the position of the forskolin binding site is indicated with a dashed circle. b A similar view of the AC9–C4 in complex with ATPαS. The asterisk indicates the absence of extended density. c Alignment of the soluble domains of AC9 in a complex with C4 and MANT-GTP (orange) or ATPαS (green) reveal a low RMSD of 1 Å. d Although the conformation of the catalytic domain is very similar, the pose of the nucleotide is different based on the density (as in (a, b)), with MANT-group of MANT-GTP pointing towards the forskolin site. e The density map corresponding to the bound MANT-GTP molecule in the AC9₁₂₅₀–Gαs–M structure (forskolin-free). The density is similar to that in (a). f The density corresponding to MANT-GTP in the AC9₁₂₅₀–Gαs–MF structure is shown as black mesh. The molecule of forskolin (Fsk) is shown as magenta sticks.

Fig. 4. Structural transitions corresponding to the distinct activator-bound states of AC9.

a The AC9–M structure (white) was structurally aligned to AC9–Gαs (orange) using the C2a domain as an anchor. The helices α4, α2′ and α3′ are indicated in the panel. The position of Gαs protein binding is indicated with a black circle. b–d Same as (a), with the AC9 bound to DARPin C4 (b; magenta), AC9₁₂₅₀–Gαs–M (c; pink), and AC9₁₂₅₀–Gαs–MF (d; blue). e Comparison of the five available structures reveals relative displacement of the active site residues, D399, D443, R487, and K1233, as well as the Q522 residue in the helix α4 adjacent to the forskolin site. The rearrangement of the active site residues proceeds in a manner that correlates with the activation state of the protein (additionally illustrated by the morphs in Movie S3). Inset: the values of Cα atom displacement from the AC9–M (white ribbon) to AC9₁₂₅₀–Gαs–MF (fully activated state, blue ribbon) are indicated in gray boxes (Å).

Fig. 5. Conformations observed in the available AC9 structures.

Based on the available structures, the G protein-free state (nucleotide-bound state, corresponding to AC9–M) can transition to a partially active state upon Gαs binding (Gαs-bound state); subsequent binding of forskolin (Fsk) fully activates AC9 (Gαs/Fsk-bound state). The G protein-activated states can be inhibited by the C2b domain of AC9 with formation of the occluded state. A partially active state can be formed upon DARPin C4 binding. This conformation of AC9 corresponds to a partially activated state, but it is a state that does not favor the formation of the stable occluded conformation, based on the structural evidence. Circles labeled “N”, “G” and “F” correspond to ATP or an ATP substitute (MANT-GTP or ATPαS), GTP and forskolin, respectively. The dark-blue rectangle corresponds to the helix α4, adjacent to the forskolin binding site. The stop-arrows indicate inability of the nucleotide and/or forskolin to bind to the AC in the occluded state.