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The relaxin receptor RXFP1 signals through a mechanism of autoinhibition

Nature Chemical Biology volume 19, pages 1013–1021 (2023)Cite this article

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Abstract

The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of a low-density lipoprotein receptor class A (LDLa) module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. In the present study, we determine the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.

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Fig. 1: Cryo-EM map and model of the RXFP1–Gs complex.
Fig. 2: Regulation of receptor signaling by ECL2 and the ectodomain.
Fig. 3: MD of RXFP1 starting from the inactive-state AlphaFold2 model.
Fig. 4: Cryo-EM and crosslinking MS reveal interactions between relaxin-2 and the leucine-rich repeats.
Fig. 5: Model of RXFP1 activation by relaxin-2.

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Data availability

The cryo-EM model and maps for RXFP1–Gs–TM are deposited under accession nos. 7TMW (PDB) and EMDB-26003, respectively. The cryo-EM map for RXFP1–Gs–FL is deposited under the accession no. EMDB-26004. The rigid-body docking model of the RXFP1 LRRs bound to relaxin-2 is available on the website for the Kruse lab at Harvard Medical School: https://kruse.hms.harvard.edu/data. The UniRef100 database and UniProt canonical sequence for RXFP1 (Q9HBX9) used for EC analysis can be found at https://www.uniprot.org. Data in Supplementary Fig. 1 are available in the Supplementary Data. Source data are provided with this paper.

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Acknowledgements

We thank M. Bao for critical reading of the manuscript and the SBGrid Consortium for computational support of structural biology software. Cryo-EM data were collected at the Harvard Center for Cryo-Electron Microscopy, Harvard Medical School, and we thank them for their support and advice during data collection. This work was funded by the National Institutes of Health (NIH) Ruth L. Kirschstein Predoctoral fellowship (no. F31GM128233 awarded to S.C.E.), Damon Runyon Postdoctoral Fellowship (no. DRG: 2489-23 awarded to J.O.-O.), a Blavatnik Biomedical Accelerator grant from Harvard Medical School (to A.C.K.) and NIH grants (nos. R01AR079489 to A.C.K. and R01CA260415 to A.C.K. and D.S.M.). MD simulations were performed using high-performance computing resources from GENCI-TGCC France (grant nos. 2021-2022 Spe00015 and A0100712461).

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Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Sarah C. Erlandson, Shaun Rawson, James Osei-Owusu, Xinyue Liu, Joao A. Paulo, Julian Mintseris, Steven P. Gygi & Andrew C. Kruse

  2. Department of Systems Biology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Kelly P. Brock & Debora S. Marks

  3. Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, Montpellier, France

    Xiaojing Cong

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Contributions

S.C.E. performed the molecular cloning, protein expression and purification, and cryo-EM grid preparation, with supervision from A.C.K. S.C.E. and J.O.-O. performed the flow cytometry and cell-signaling assays, with supervision by A.C.K. S.C.E. and S.R. processed the cryo-EM data. S.C.E. performed model building and refinement, with supervision from A.C.K. K.P.B. performed the EC analysis, with supervision from D.S.M. X.L., J.A.P. and J.M. performed the crosslinking MS, with supervision from S.P.G. X.C. performed the molecular dynamics simulations. S.C.E., X.C. and A.C.K. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Andrew C. Kruse.

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Competing interests

A.C.K. and S.C.E. are inventors on patent application PCT/US2021/031260 for engineered single-chain relaxin proteins. A.C.K. is a cofounder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a nonprofit research institute. D.S.M. is a cofounder of Seismic Therapeutic, a consultant for Tectonic Therapeutic, Dyno Therapeutics, Jura Bio and Octant Bio, and a venture partner at Catalio Capital Management. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Wenqing Shui, Jin-Peng Sun and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Engineering and purification of the RXFP1–Gs complex.

a, Diagram of the primary structure of RXFP1 domains versus the RXFP1-miniGs399-20res fusion construct. b, Flow cytometry cell surface expression tests in Expi293F tetR cells for RXFP1-miniGs fusion constructs. Data is mean ± s.e.m., n = 3 technical replicates. c, Gs signaling assay comparing the signaling levels of wild type RXFP1 versus RXFP1-miniGs399-20res in response to relaxin-2. Data is mean ± s.e.m., n = 3 technical replicates. d, Size exclusion chromatography profile for the RXFP1–Gs complex. Arrow indicates the peak fractions pooled for RXFP1–Gs. e, Coomassie-stained SDS-PAGE gel for the RXFP1–Gs complex (Representative from 8 purification gels). f, Flow cytometry competition binding assay for SE00111. SE001 competes with 200 nM SE301 for binding to wild type RXFP1. The Ki for SE001 was calculated to be 3.9 nM; data is mean ± s.e.m., n = 3 technical replicates.

Source data

Extended Data Fig. 2 Cryo-EM data processing for the 7TM domain of RXFP1–Gs.

a, Cryo-EM data processing scheme for the 7TM domain of RXFP1 in complex with Gs. Shown are representative processing steps for one of four individual datasets and the steps used for the combined datasets. b, Representative micrograph from the RXFP1–Gs complex datasets (Scale bar = 50 nm; from 13,457 micrographs). c, Two-dimensional class averages for the 7TM domain of RXFP1 and G proteins. d, Angular distribution of particles in the final refinement for the 7TM domain with G proteins. e, Fourier shell correlation (FSC) used to determine the overall map resolution. f, Map to model FSC curve. g, cryoSPARC non-uniform refinement map colored by local resolution.

Extended Data Fig. 3 Cryo-EM data processing for the full−length RXFP1–Gs complex.

a, Cryo-EM data processing scheme for full-length RXFP1 in complex with Gs. Shown are representative processing steps for one of four individual datasets and the steps used for the combined datasets. b, Two-dimensional class averages for the full-length RXFP1–Gs complex. c, FSC used to determine the overall resolution of the map. d, RELION map of the full-length RXFP1 complex colored by local resolution at two different contour levels to display both the TM helices and receptor ectodomain.

Extended Data Fig. 4 Alignments of RXFP1’s ECL2 with GPR52 and family A orthosteric agonists.

Alignment of active-state RXFP1 (green, with ECL2 in magenta) (a), the β2 adrenergic receptor (gray) bound to adrenaline (purple; PDB ID: 4LDO)16 (b), and the angiotensin II type I receptor (gray) bound to the angiotensin II analog S1I8 (purple; PDB ID: 6DO1)17 (c), with GPR52 (gray with ECL2 in purple; PDB ID: 6LI3)18.

Extended Data Fig. 5 Cell surface expression and SE301 binding for RXFP1 constructs.

a-d, Flow cytometry cell surface expression tests with HEK293T cells for RXFP1 ECL2 mutants (a), Leu402 and Leu403 hinge region mutants (b), ectodomain truncation constructs (c), and evolutionary coupling analysis Ile396 and Ser397 hinge mutants (d). Data is mean ± s.e.m., n = 3 technical replicates. Cell surface expression was calculated as a percentage of wild type RXFP1 expression level. e-f, Ratio of SE301 (Fc-tagged relaxin-2) binding to receptor expression for flow cytometry binding assays in Expi293F cells11. The ratio is calculated by dividing the mean fluorescence intensity (MFI) of SE301 binding by the MFI of the expression level. Data is mean ± s.e.m., n = 3 technical replicates. Deletion or mutations to the linker region reduce the ratio of binding to expression, while LDLa deletions and ECL2 mutations retain an ability to bind SE301 (e). Ectodomain deletions (7TM + β2N-term) reduce the ratio of binding to expression, while mutations to the hinge region maintain an ability to bind SE301 (f).

Source data

Extended Data Fig. 6 Evolutionary coupling analysis of RXFP1.

a, Evolutionary couplings for RXFP1 residues 405–689 (black) compared to the active-state structure contacts (green) show close agreement between predicted contacts from ECs and the cryo-EM model. b, Evolutionary couplings for RXFP1 residues 120–757 (black) compared to the active-state 7TM structure and LRR AlphaFold227 model contacts (blue), highlighting ECL2 evolutionary couplings that provide insight into two potential loop conformations in magenta (T559ECL2–Ile396, Gly561ECL2–Ser397, Phe564ECL2–Ile396, Phe564ECL2–Val6667.38). c, Diagram of RXFP1 domains. Stars indicate regions of RXFP1 containing residues with the highest scoring ECs with ECL2, TM7 and the hinge region. d, The Phe564ECL2 and Val6667.38 residues from evolutionary coupling analysis are in close contact in the RXFP1 active-state structure.

Extended Data Fig. 7 Molecular dynamics of RXFP1 7TM deactivation.

a, The truncated 7TM domain is deactivated by adding a sodium in the conserved sodium-binding site. This leads to an inactive state during the simulations that closely resembles the AlphaFold2 model. b-c, Ionic lock distance and Cα RMSD of the TM region with respect to the AlphaFold2 model. Shown here are probability density distributions during 50 ns of REST2-MD simulations (10 frames/ns, n = 500). SE, integrated squared error between the density estimate and a standard normal density function. Vertical dashed lines indicated the values in the AlphaFold2 model and in the initial model based on cryo-EM.

Extended Data Fig. 8 Principal component analysis.

Principal component analysis of halfLRRs-7TM WT, S397A, and S397A/L402A/L403A are presented together. a, b, Projection of the simulation frames onto the 2D and 3D spaces defined by the 3 largest global PCs, covering 38.58%, 22.03% and 11.17% of the total variance, respectively. Dashed circle indicates (roughly) the active-like conformations of halfLRRs-7TM S397A. c, Conformational variance associated with PC3, reflecting TM6 opening of the S397A mutant upon activation. The same PC was found in the analysis of the S397A mutant alone (PC2 in Fig. 3d). For clarity, only 50 frames per system (1 frame/ns) are shown here.

Extended Data Fig. 9 Comparison of LHCGR and RXFP1 structures.

a, b, The hybrid model of active-state RXFP1 (based on our 7TM domain structure, cryo-EM maps, and AlphaFold2 model of the LRRs with docked relaxin-2 hormone) compared to active-state LHCGR (PDB ID: 7FIG) and inactive-state LHCGR (PDB ID: 7FIJ)8. The receptors are aligned on the 7TM domain. b, c, Side views (b) and top view (c) of the hybrid model of active-state RXFP1 compared to active-state LHCGR (PDB ID: 7FIG). The receptors are aligned on the 7TM domain and the ligands (relaxin-2 and chorionic gonadotropin) not displayed in order to highlight differences in active-state LRR orientations.

Extended Data Fig. 10 Signaling of RXFP1 constructs by the small molecule agonist ML290.

a, Gs signaling at wild type RXFP1 and ECL2 mutants at basal levels and in response to 490 nM ML290. Data is mean ± s.e.m., n = 3 technical replicates. b, Gs signaling at constructs of the RXFP1 7TM domain fused to the β2 adrenergic receptor N-terminus at basal levels and in response to 490 nM ML290. Data is mean ± s.e.m., n = 3 technical replicates.

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Supplementary Information

Supplementary Figs. 1–5, Tables 1–12 and legend for Supplementary Video 1.

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Supplementary Data

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Supplementary Video 1

Supplementary Video 1, related to Fig. 4 and Extended Data Fig. 3 Dynamics of RXFP1’s active-state ectodomain. Continuous heterogeneity within the final cryo-EM particle stack used for refinement of the full-length RXFP1–Gs complex. Flexibility of the ectodomain with respect to the 7TM domain and G proteins was visualized using 3D variability analysis in cryoSPARC2.

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Erlandson, S.C., Rawson, S., Osei-Owusu, J. et al. The relaxin receptor RXFP1 signals through a mechanism of autoinhibition. Nat Chem Biol 19, 1013–1021 (2023). https://doi.org/10.1038/s41589-023-01321-6

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