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Structure of adenylyl cyclase 5 in complex with Gβγ offers insights into ADCY5-related dyskinesia

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

The nine different membrane-anchored adenylyl cyclase isoforms (AC1–9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gβγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gβγ and a dimeric form of AC5 that could be involved in its regulation. Gβγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gβγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gβγ and show reduced conditional activation by Gβγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gβγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

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Fig. 1: Characterization of AC5 and its complex with Gβγ.
Fig. 2: The AC5–Gβγ interface.
Fig. 3: The C1b-α2 and H121023–1034 regions in AC5 strongly contribute to Gβγ-mediated AC5 activation.
Fig. 4: Location of disease-associated AC5 mutations and impaired responses to Gβγ of those associated with familial dyskinesia.
Fig. 5: Homodimerization of AC5 could be linked to regulation by Gβγ.
Fig. 6: Proposed model for Gβγ-mediated AC5 activation.

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Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The structures of the AC5–Gβγ complex and the AC5 homodimer have been deposited into the Protein Data Bank (PDB) under accession nos. 8SL3 and 8SL4 and the Electron Microscopy Data Bank (EMDB) under accession nos. EMDB-40572 and EMDB-40573, respectively. Requests for the plasmids and other reagents should be submitted to the corresponding author, J.J.G.T. Source data are provided with this paper.

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Acknowledgements

We thank the Purdue Life Sciences Electron Microscopy Facility from the Institute for Cancer Research (National institutes of Health (NIH) grant no. P30 CA023168) and from the Indiana Clinical and Translational Sciences Institute (CTSI) FY21 CTSI PostDoc Challenge for their support. This work was supported by NIH grants (nos. CA254402, CA221289, CA023168 and HL071818 to J.J.G.T., GM60419 and GM145921 to C.W.D., and DA051876, NS119917 and NS111070 to V.J.W.), the Walther Cancer Foundation (J.J.G.T.) and American Heart Association Postdoctoral fellowship 903842 (Y.-C.Y.).

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Yu-Chen Yen, Chun-Liang Chen & John J. G. Tesmer

  2. Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA

    Yong Li & Carmen W. Dessauer

  3. Purdue Cryo-EM Facility, Hockmeyer Hall for Structural Biology, Purdue University, West Lafayette, IN, USA

    Thomas Klose

  4. Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA

    Val J. Watts & John J. G. Tesmer

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Contributions

J.J.G.T., V.J.W., C.W.D. and Y.-C.Y. conceived the project and provided the methodology. Y.-C.Y., Y.L., C.-L.C. and T.K. carried out the investigations. J.J.G.T., V.J.W. and C.W.D. acquired funding, and administered and supervised the project. Y.-C.Y. wrote the original draft of the manuscript. J.J.G.T., V.J.W., C.W.D., Y.-C.Y., Y.L., C.-L.C. and T.K. reviewed and edited the manuscript.

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Correspondence to John J. G. Tesmer.

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Nature Structural & Molecular Biology thanks Kirill Martemyanov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Purification and characterization of AC5.

(A) AC5 was purified using anti-FLAG M2 affinity resin and analyzed by Coomassie blue stained SDS-PAGE. The AC activity of purified AC5 was determined in the presence of 10 mM MgCl2, 0.5 mM ATP at the indicated concentrations of (B) forskolin or (C) Gαs·GTPγS. The data shown are the mean ± SD from three independent assays.

Source data

Extended Data Fig. 2 Activation of AC5 by Gβγ and purification of an AC5–Gβγ complex.

(A) AC activity of purified AC5 was determined in the presence of 10 mM MgCl2 and 0.5 mM ATP with or without 250 nM Gαs·GTPγS, in the absence or presence of 400 nM Gβ1γ2. Data presented are the mean ± SD. Statistical significance was assessed using an unpaired Welch’s t-test (two-sided) across at least five independent experiments. (B) The AC5–Gβγ complex was purified using anti-FLAG M2 affinity resin and injected onto a Superose 6 Increase 3.2/300 column equilibrated with 50 mM HEPES pH 8, 100 mM NaCl, 10 mM MgCl2, and 0.01 % LMNG. Peak fractions highlighted in pink were pooled, concentrated, and (C) analyzed by SDS-PAGE (note that Gγ2 runs off the gel).

Source data

Extended Data Fig. 3 Cryo-EM data processing workflow and resolution analysis of the AC5–Gβγ complex.

The workflow, including a representative micrograph, 2D class averages (box size = 256 Å, mask = 180 Å), local resolution map, and Fourier shell correlation (FSC) curves calculated from two independent reconstructions by CryoSPARC. The nominal resolution of the resulting map, as defined by the 0.143 cutoff, is indicated by the horizontal blue line.

Extended Data Fig. 4 Binding mode of the AC5–Gβγ complex predicted by AlphaFold Multimer and comparison with other Gβγ complexes.

(A) The docked model of AC5–Gβγ complex determined in this study (left, PDB entry 8SL3) and that predicted by AlphaFold Multimer (right). For comparison, some other Gβγ complexes are shown, including (B) Gα (PDB entry 1GOT), (C) GRK2 (PDB entry 6U7C), and (D) Prex-1 (PDB entry 6PCV) in complex with Gβγ. The red highlighted region in each structure contacts the central core ‘hotspot’ of Gβ close to or centered at Trp99.

Extended Data Fig. 5 Purified AC5 and Gβγ variants used in this study.

The purity of AC5 and Gβγ variants was assessed using Coomassie blue stained SDS-PAGE (Gγ at 7.5 kDa runs off the gel). With the exception of AC5H12swap and GβR52Eγ, all other mutants were from at least two distinct preps and exhibit comparable levels of purity.

Source data

Extended Data Fig. 6 Cryo-EM analysis of various AC5 samples described in this manuscript.

Representative micrographs and 2D class averages are shown for each sample. Note that homodimers (yellow boxes) are only observed in GDN regardless of activation status.

Source data

Extended Data Fig. 7 Sample preparation and cryo-EM data processing workflow of the AC5–Gβγ–Gαs complex.

A presumptive AC5–Gβγ–Gαs complex was purified using Anti-FLAG antibody pulldown and analyzed by SDS-PAGE with Coomassie blue staining (top left). Cryo-EM data processing workflow includes a representative micrograph, 2D class averages (box size = 324 Å, mask = 200 Å for AC5–Gβγ on the left and 180 Å for dimeric AC5 on the right), and reconstructed 3D models. The dataset reveals two distinct populations, with one corresponding to the AC5–Gβγ monomer (bottom left) and the other an AC5 dimer containing density corresponding to Gαs subunits (bottom right). Below the dimeric AC5 map, the central slice is displayed in two directions. Note that the 3×4 array of TM helices line up in the slice on the right into three vertical lines in each subunit, consistent with the above cartoon. The scale bar of the heat map indicates arbitrary units of density obtained from the particle images.

Extended Data Fig. 8 Characterization of Purified AC5 in LMNG and GDN micelles.

(A) Coomassie blue stained SDS-PAGE showing purified AC5 reconstituted in either LMNG or GDN. (B) AC activity (mean ± SD) of purified AC5 reconstituted in LMNG or GDN was determined in the presence of 10 mM MgCl2 and 0.5 mM ATP. Statistical significance was assessed using an unpaired Welch’s t-test (two-sided) across six independent experiments. (C) Gαs and (D) Gβγ dose-response curve for AC5 reconstituted in LMNG or GDN. AC5 activity was determined in the presence of 10 mM MgCl2, 0.5 mM ATP at the indicated Gαs concentration or 100 nM Gαs·GTPγS at the indicated Gβγ concentrations, with data presented as mean ± SD from at least two independent assays, each consisting of duplicates.

Source data

Extended Data Fig. 9 Sequence alignment of the C1b region.

Residue similarities are colored according to the BLOSUM62 score. Secondary structures of the primary sequence for the AC5 structure here are displayed on top, and the AlphaFold2 predicted model for the C1b region in AC5 is shown at the bottom as a reference for the named helices. The predicted ‘PFAHL’ Gβγ binding site in AC2 is highlighted in an orange box. Residues modelled in contact with Gβγ in the AC5–Gβγ complex (PDB entry 8SL3) are drawn in red color.

Extended Data Fig. 10 Comparison of mAC structural features.

(A) Domain architecture of mAC isoforms. (B) Sequence alignment of the N-α3 helix among mAC isoforms, with secondary structure from AC5 indicated above. (C) Comparison of a predicted binding pocket in the extracellular domain of AC5 and AC8, with the residues predicted to form the binding pocket highlighted in red in both the cartoon (left) and surface (middle) representations. Additionally, the electrostatic potential is shown (right), highlighting the negatively charged surface of the extracellular domain. (D) Superposition of AC5 (PDB entry 8SL3) and AC9 (PDB entry 6R3Q), revealing similar organization of the 12 TM helices (with the extracellular domain of AC5 removed for clarity, left) but subtle differences in the coiled-coil domain (right). (E) Overlay of AC5 (PDB entry 8SL3) and AC9 (PDB entry 6R3Q) suggests the potential simultaneous binding of Gαs and Gβγ to the catalytic core. (F) AlphaFold multimer prediction of the AC5–Gαs–Gβγ complex. The C1b-α2 helix is highlighted in red in both (E) and (F).

Supplementary information

Source data

Source Data Extended Data Figs. 1, 2, 5, 7 and 8

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Source Data Fig. 3

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Source Data Fig. 4

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Source Data Extended Data Fig. 1

Source data used for generating the graph.

Source Data Extended Data Fig. 2

Source data used for generating the graph.

Source Data Extended Data Fig. 8

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Yen, YC., Li, Y., Chen, CL. et al. Structure of adenylyl cyclase 5 in complex with Gβγ offers insights into ADCY5-related dyskinesia. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01263-0

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