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Crystal structure of the human σ1 receptor

Nature volume 532, pages 527–530 (2016)Cite this article

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Abstract

The human σ1 receptor is an enigmatic endoplasmic-reticulum-resident transmembrane protein implicated in a variety of disorders including depression, drug addiction, and neuropathic pain1. Recently, an additional connection to amyotrophic lateral sclerosis has emerged from studies of human genetics and mouse models2. Unlike many transmembrane receptors that belong to large, extensively studied families such as G-protein-coupled receptors or ligand-gated ion channels, the σ1 receptor is an evolutionary isolate with no discernible similarity to any other human protein. Despite its increasingly clear importance in human physiology and disease, the molecular architecture of the σ1 receptor and its regulation by drug-like compounds remain poorly defined. Here we report crystal structures of the human σ1 receptor in complex with two chemically divergent ligands, PD144418 and 4-IBP. The structures reveal a trimeric architecture with a single transmembrane domain in each protomer. The carboxy-terminal domain of the receptor shows an extensive flat, hydrophobic membrane-proximal surface, suggesting an intimate association with the cytosolic surface of the endoplasmic reticulum membrane in cells. This domain includes a cupin-like β-barrel with the ligand-binding site buried at its centre. This large, hydrophobic ligand-binding cavity shows remarkable plasticity in ligand recognition, binding the two ligands in similar positions despite dissimilar chemical structures. Taken together, these results reveal the overall architecture, oligomerization state, and molecular basis for ligand recognition by this important but poorly understood protein.

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Figure 1: Overall structure of the σ1 receptor.
Figure 2: Structure of the σ1 protomer.
Figure 3: Ligand recognition.

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Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the σ1 receptor bound to PD144418 and 4-IBP have been deposited in the Protein Data Bank under accession numbers 5HK1 and 5HK2, respectively.

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Acknowledgements

We thank beamline staff at the Advanced Photon Source for their technical assistance and support. We thank S. Blacklow and B. Zimmerman for discussions and input throughout the project, and K. Arnett for assistance with SEC–MALS experiments. Financial support for this work was provided by departmental startup funds. S.Z. is supported by a Merck Postdoctoral Fellowship in Biological Chemistry and Molecular Pharmacology, and H.R.S. is supported by the National Institutes of Health Cellular and Developmental Biology training grant at Harvard Medical School (T32GM007226).

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Author notes

  1. Hayden R. Schmidt and Sanduo Zheng: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Hayden R. Schmidt, Sanduo Zheng, Esin Gurpinar & Andrew C. Kruse

  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Antoine Koehl & Aashish Manglik

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Contributions

Receptor purification and crystallization experiments were conducted by H.R.S., E.G., and A.C.K. X-ray data collection was performed by H.R.S., A.M., A.K., and A.C.K. Data processing, phase calculation, and structure refinement were performed jointly by H.R.S., S.Z., and A.C.K. SEC–MALS experiments were performed by A.C.K., radioligand binding by H.R.S., and native-PAGE by S.Z. Overall project design, molecular cloning, and pilot studies were conducted by A.M. and A.C.K.

Corresponding author

Correspondence to Andrew C. Kruse.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Assessment of σ1 functional properties and biochemical quality.

a, Saturation binding curve to measure Kd for 3H (+)-pentazocine, with points shown as mean ± s.e.m. b, Competition binding measurement of affinities for the two co-crystallized ligands with points shown as mean ± s.e.m. c, Summary of binding affinities with 95% confidence intervals for Kd/Ki values. d, Analysis of receptor purity by SDS–PAGE. e, Analytical size exclusion of purified σ1 receptor in LMNG/CHS detergent buffer on a Superdex 200 column.

Extended Data Figure 2 Representative electron density.

a, Composite omit 2Fo − Fc electron density contoured at 1.0σ for σ1 receptor bound to PD144418, showing a loop from Val73 to Glu78 as well as surrounding residues. b, The same map over a loop from His116 to Ser125. c, The equivalent map to that in a, calculated for σ1 receptor bound to 4-IBP. d, The equivalent map to that in b, calculated for σ1 receptor bound to 4-IBP.

Extended Data Figure 3 Lattice contacts.

a, Lattice packing of the σ1 receptor viewed parallel to the membrane plane. b, c A view normal to the membrane and another parallel view, respectively. d, A single σ1 trimer is shown, with lattice contact residues highlighted in magenta sticks. Lattice contacts are formed primarily through interactions of the relatively poorly conserved transmembrane helices.

Extended Data Figure 4 Hydrophobicity analysis.

a, The structure of σ1 receptor shows a hydrophilic (blue) surface on the cytosolic face (left), while transmembrane domains and the membrane-facing surface of the receptor trimer are hydrophobic (orange; right panels). Hydrophobicity analysis was conducted using UCSF Chimera.

Extended Data Figure 5 Sequence conservation.

The results of an alignment of 277 sigma receptor sequences from vertebrates with Homo sapiens, Mus musculus, Danio rerio, and Xenopus laevis displayed. Residues with 98%, 80%, and 60% similarity are shown in black, grey, and light grey respectively. Secondary structure elements are shown above the alignment on the basis of the human σ1 receptor crystal structure. Open black circles mark residues within 4 Å of the ligand-binding site, solid black circles below the alignment denote residues located in the trimerization interface, and a red circle marks the site of the E102Q mutation associated with amyotrophic lateral sclerosis.

Extended Data Figure 6 Trimerization interface.

a, b, Two views of the trimerization interface are shown, coloured by sequence conservation. Residues highlighted in yellow are more than 80% conserved among a selection of 300 σ1 receptor homologues, and residues in orange surface are more than 98% conserved. c, d, Close-up views of the interface, showing the extensive hydrophobic and polar contacts at the oligomerization interface.

Extended Data Figure 7 Omit maps of PD144418 and 4-IBP.

a, An Fo − Fc omit map contoured at 1σ showing the electron density (purple) of PD144418 (yellow). b, An equivalent map showing the electron density (purple) of 4-IBP (orange).

Extended Data Figure 8 Oligomerization state.

a, Analysis of receptor oligomerization by SEC–MALS in the presence of the classical antagonist NE-100 or (b) the classical agonist SKF-10,047. The peak is 38% detergent and 62% protein by mass. The total mass of each component varies throughout the peak, indicating a mix of oligomeric species. c, Analysis of oligomerization state by blue native PAGE (left) and a higher-resolution detergent-supplemented tris-glycine native PAGE gel (right), showing a similar polydisperse profile. Discrete oligomers are marked with red dots, corresponding to possible trimers, hexamers, and higher-order species. d, In a mixed micelle of lauryl maltose neopentyl glycol and cholesterol hemisuccinate, modest differences in SEC profile are observed between agonist- and antagonist-treated receptor.

Extended Data Table 1 Data collection and refinement statistics
Full size table
Extended Data Table 2 Structural homologues of the σ1 receptor
Full size table

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Schmidt, H., Zheng, S., Gurpinar, E. et al. Crystal structure of the human σ1 receptor. Nature 532, 527–530 (2016). https://doi.org/10.1038/nature17391

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