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. 2022 Aug 18;11:e77032. doi: 10.7554/eLife.77032

Figure 1. Structure of Cya–NB4 complex.

(A) Schematic representation of the mammalian membrane adenylyl cyclases (ACs), indicating the key elements of AC structure: 12 transmembrane (TM) domains, 2 catalytic domains, an ATP, and a forskolin (Fsk)-binding site. The protein is depicted in a G-protein-bound state. (B, C) A schematic representation of Rv1625c/Cya, illustrating the regions resolved in the cryo-EM structure. The TM region is coloured orange, the helical domain (HD) is green, the catalytic domain is blue. Regions absent in the cryo-EM structure are grey. (D) The activity of the full-length Cya in detergent is similar in the absence (yellow) and in the presence of nanobody NB4 (pink); the soluble domain of Cya (SOL, blue) shows low levels of activity. The activity of SOL in the presence of NB4 (cyan) is similar to SOL alone. For all experiments, the data are shown as mean ± standard error of the mean (SEM) (n = 3; for SOL, n = 6). (E) The density map of Cya–NB4 complex at 3.57 Å resolution, obtained using masked refinement of the best dataset with C2 symmetry imposed. (F) The corresponding views of the atomic model of Cya–NB4 complex, coloured as in B, C. ‘N’ indicates the N-terminal part of the protein; ‘HD’ – helical domain; ‘CAT’ – catalytic domain.

Figure 1—source data 1. Adenylyl cyclase acivity data and analysis (Figure 1D).

Figure 1.

Figure 1—figure supplement 1. Purification and characterization of Cya.

Figure 1—figure supplement 1.

(A) Size-exclusion chromatography (SEC) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the purified Cya. SEC was peformed using a Superose 6 Increase column. (B, C) Same as in A, for NB4 (B) and for Cya-SOL. SEC was performed using a Superdex 200 Increase column. (D) Analytical SEC analysis Cya–NB4 interaction was performed using the Agilent Bio SEC-5 column. The shift of the SEC peak upon NB4 binding is indicated with an arrow; a dashed line indicates the position of the Cya alone. (E) Isothermal titration calorimetry (ITC) analysis of Cya-SOL/NB4 binding; mean Kd ± standard deviation (SD) is indicated in the graph (n = 3). ITC thermogram integration was carried out using NITPIC. Global analysis of integrated thermograms was performed using SEDPHAT and figures were generated using GUSSI.

Figure 1—figure supplement 2. Cryo-EM processing workflow.

Figure 1—figure supplement 2.

(A) A processing pipeline for 3D reconstruction of the Cya–NB4 complex. Three datasets were processed in both C1 and C2 symmtery in parallel. Particles resulting from the best 3D classes were pooled, duplicates removed, and the resulting particle selection was refined in C2 symmetry for each dataset. All three datasets were combined and further processed by particle polishing and CTF refinement, resulting in two density maps: one processed in C1 symmetry (at a resolution of 3.83 Å) and another one in C2 symmtery (at a resolution of 3.57 Å), as detailed in ‘Materials and methods’. (B) An example of a cryo-EM micrograph the bar corresponds to 200 Å. (C) Representative 2D classes reveal distinct views of the Cya–NB4 complex.

Figure 1—figure supplement 3. Properties of the Cya–NB4 density maps.

Figure 1—figure supplement 3.

(A) Local resolution, angular distribution, and Fourier shell correlation (FSC) curve of the Cya–NB4 complex processed in C1 symmetry. Scale bar indicates local resolution range in Å. (B) Isolated cryo-EM density for each of TM helix and the helical domain (HD) contoured at 8σ (top). Isolated cryo-EM density for catalytic domain and NB4 contoured at 8σ, with extracellular NB4 contoured with 4σ (bottom). (C, D) Same as A, B, for the Cya–NB4 processed in C2 symmetry. All density map features are contoured at 8σ.

Figure 1—figure supplement 4. Cryo-EM density map and model of Cya in C1 symmetry.

Figure 1—figure supplement 4.

(A) The density map of Cya–NB4 complex at 3.83 Å resolution, obtained using masked refinement of the best dataset with C1 symmetry. (B) The corresponding views of the atomic model of Cya–NB4 complex, coloured as in B, C. ‘N’ indicates the N-terminal part of the protein; ‘HD’ – helical domain; ‘CAT’ – catalytic domain.

Figure 1—figure supplement 5. X-ray structure of the catalytic domain of Cya, Cya-SOL, bound to NB4.

Figure 1—figure supplement 5.

(A) Illustration of a crystallographic unit cell in the crystals of catalytic domain of Cya–NB4 complex showing the arrangement of one copy of Cya-SOL–NB4 complex (red) in an asymmetric unit (red). Analysis of the interaction between Cya-SOL–NB4 with its symmetry mates (grey) shows that Cya-SOL–NB4 does engages in non-native interactions with its neighbours, consistent with formation of a crystallographic dimer (right). (B) The surface representations of the catalytic domain of Cya (right) and NB4 (left), coloured according to the calculated electrostatic potential, suggest a role of the surface-exposed charged residues in the Cya–NB4 interaction. (C) Fo–Fc omit map of MANT-GTP (left) bound to Cya-SOL. Density is contoured at 3σ, carved to 1.6 Å in Pymol. (D) Despite retaining the ability to bind the MANT-GTP molecule via one-half of its catalytic site, the position of the nucleotide is distinct from that observed in the structure of the nucleotide-bound dimeric Cya.