Abstract
The heart, in addition to its primary role in blood circulation, functions as an endocrine organ by producing cardiac hormone natriuretic peptides. These hormones regulate blood pressure through the single-pass transmembrane receptor guanylyl cyclase A (GC-A), also known as natriuretic peptide receptor 1. The binding of the peptide hormones to the extracellular ___domain of the receptor activates the intracellular guanylyl cyclase ___domain of the receptor to produce the second messenger cyclic guanosine monophosphate. Despite their importance, the detailed architecture and ___domain interactions within full-length GC-A remain elusive. Here we present cryo-electron microscopy structures, functional analyses and molecular dynamics simulations of full-length human GC-A, in both the absence and the presence of atrial natriuretic peptide. The data reveal the architecture of full-length GC-A, highlighting the spatial arrangement of its various functional domains. This insight is crucial for understanding how different parts of the receptor interact and coordinate during activation. The study elucidates the molecular basis of how extracellular signals are transduced across the membrane to activate the intracellular guanylyl cyclase ___domain.
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Data availability
The cryo-EM density maps and corresponding coordinates were deposited to the EMDB and PDB, respectively, under the following accession codes: EMD-44430 (apo full-length GC-A); EMD-44432 and PDB 9BCO (apo ICD); EMD-44433 and PDB 9BCP (apo KHD); EMD-44429 and PDB 9BCL (apo ECD (state 1)); EMD-44431 and PDB 9BCN (apo ECD (state 2)); EMD-44437 (active full-length GC-A); EMD-44436 and PDB 9BCS (active ICD); EMD-44440 and PDB 9BCV (active GCD); EMD-44434 and PDB 9BCQ (ANP-bound ECD). Source data are provided with this paper.
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Acknowledgements
We thank L. Levin, J. Levitz, T. Maack, J. Meyerson and members of our research groups for helpful discussion and comments on the paper and the Antibody and Bioresource Core Facility at the Memorial Sloan Kettering Cancer Center for help with the monoclonal antibody generation. This work was supported by National Institutes of Health (NIH) grant GM138676 (X.Y.H), NIH–National Cancer Instute Cancer Center Support Grant P30 CA008748 (R.K.H) and the startup funding project 27110 at the University of North Carolina, Chapel Hill (Y.M.). The Simons EM Center and the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center are supported by grants from the NIH National Institute of General Medical Sciences (GM103310), NYSTAR and the Simons Foundation (SF349247). Supercomputing resources were used with allocation award TG-MCB180049 through the Extreme Science and Engineering Discovery Environment, which is supported by National Science Foundation grant ACI-1548562 and project M2874 through the National Energy Research Scientific Computing Center.
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S.L. expressed and purified GC-A, made cryo-EM grids and performed cryo-EM screening, data collection, structure determination, model building and paper preparation. A.M.P. and N.P. performed initial cryo-EM screening and density map determination of the ECD under the supervision of R.K.H. J.W. performed and analyzed MD simulations under the supervision of Y.M. L.Z. performed cGMP assays under the supervision of W.L. E.E. helped with cryo-EM data collection. X.Y.H. supervised the project, interpreted the data and wrote the paper. All authors contributed toward the final version of the paper.
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Nature Structural & Molecular Biology thanks Michaela Kuhn 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 functional characterization of the human GC-A.
a, Size-exclusion chromatography profile of GC-A after elution from immobilized GFP-nanobody beads. b, SDS-PAGE results of peak fraction from the GC-A dimer. c, d, cGMP assays of the full-length wild-type and mutant human GC-As expressed in CHO cells. The GC-A mutants are located in the KHD and include two groups: one group is in the long extended loop region (L475G/W476G/V478G/W480G/V483G, E489G/R490G/H491G/R493G, and ΔE489-F521), and the other group includes residues involved in ATP interaction (N628A, K630A, N633A, K535A, D646A, and K666A). Data are represented as mean ± SD of three experiments.
Extended Data Fig. 2 Flow chart of Cryo-EM data processing for full-length GC-A.
The initial full-length GC-A map was determined using a small subset from the total micrographs that contributed to the final structural determination. A representative motion corrected micrograph is provided with circled particles in the “side view.” A few selected 2D classes are shown highlighting the orientation preference issue and poor particle-alignment issue even at low resolution ranges. The thumbnail maps post to refinement and classification jobs with dashed squares demonstrate the selected 3D classes and their qualities. Reference maps or particles fed into subsequent refinement or classification jobs are indicated by yellow, red and blue arrows.
Extended Data Fig. 3 Cryo-EM and model qualities of apo GC-A structures.
a, Particle orientation distributions are shown in heat maps. b, Fourier correlation curves of the Cryo-EM maps calculated in Cryosparc and of the map-to-model in PHENIX are shown in black and blue curves with 0.143 and 0.5 cutoffs, respectively, that determined the final resolutions. c, Local resolutions of Cryo-EM structures shown in heat maps.
Extended Data Fig. 4 Flow chart of Cryo-EM data processing for the ECD of GC-A.
a, 2D classes are shown highlighting the highly populated orientations of the particles. The thumbnail maps post to refinement and classification jobs with dashed squares demonstrate the selected 3D classes and their qualities. Reference maps or particles fed into subsequent refinement or classification jobs are indicated by yellow and red arrows. b, Cryo-EM density and models of the previously reported and putative glycosylation sites in GC-A.
Extended Data Fig. 5 Flow chart of Cryo-EM data processing for the ICD and KHD of GC-A.
a, The thumbnail maps post to refinement and classification jobs with dashed squares demonstrate the selected 3D classes and their qualities. Reference maps or particles fed into subsequent refinement or classification jobs are indicated by yellow and blue arrows. b, Cryo-EM density and models of the ATP molecules from GC-A monomers.
Extended Data Fig. 6 Flow chart of Cryo-EM data processing for ANP-bound full-length GC-A.
The initial full-length GC-A map was determined using a small subset from the total micrographs that contributed to the final structural determination. A few selected 2D classes are shown highlighting the side-view. The thumbnail maps post to refinement and classification jobs with dashed squares demonstrate the selected 3D classes and their qualities. Reference maps or particles fed into subsequent refinement or classification jobs are indicated by arrows.
Extended Data Fig. 7 Flow chart of Cryo-EM data processing for the ANP bound ECD of full-length GC-A.
The thumbnail maps post to refinement and classification jobs with dashed squares demonstrate the selected 3D classes and their qualities. Reference maps fed into subsequent refinement or classification jobs are indicated by yellow and red arrows.
Extended Data Fig. 8 Cryo-EM and model qualities of ANP bound GC-A structures.
a, Particle orientation distributions are shown in heat maps. b, Fourier correlation curves of the Cryo-EM maps calculated in Cryosparc and of the map-to-model in PHENIX are shown in black and blue curves with 0.143 and 0.5 cutoffs, respectively, that determined the final resolutions. c, Local resolutions of Cryo-EM structures shown in heat maps.
Extended Data Fig. 9 GaMD simulations of GC-A without and with bound ANP.
a, Ensemble of the top 20 conformations of apo GC-A colored by reweighted free energy values obtained from the GaMD simulations. b, Ensemble of the top 20 conformations of ANP-bound GC-A colored by reweighted free energy values obtained from the GaMD simulations. c–g, Time courses of the RMSDs of ANP (c), GTP in Chain A (d) and Chain B (e) relative to their cryo-EM conformations calculated from the GaMD simulations of the ANP-bound GC-A. 2D free energy profiles of the ANP-bound GC-A calculated from the GaMD simulations regarding the RMSDs of ANP and GTP in Chain A (f) and in Chain B (g).
Extended Data Fig. 10 Functional studies of mutant GC-As.
a, Structure of the CC ___domain and the guanylyl cyclase catalytic ___domain of GC-A. b, The helix-turn-helix motif links the CC to the cyclase ___domain. P822 is marked in red color. c, Structural comparison of the CCs in the apo and the active states of GC-A. d, e, cGMP assays of the full-length wild-type and mutant human GC-As expressed in CHO cells. ND: not determined. Data are represented as mean ± SD of three experiments.
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Liu, S., Payne, A.M., Wang, J. et al. Architecture and activation of single-pass transmembrane receptor guanylyl cyclase. Nat Struct Mol Biol 32, 469–478 (2025). https://doi.org/10.1038/s41594-024-01426-z
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DOI: https://doi.org/10.1038/s41594-024-01426-z
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