Fu C et al. (2024), Insight into binding of endogenous neurosteroid...

XB-ART-60779

Nat Commun 2024 Jul 04;151:5619. doi: 10.1038/s41467-024-49894-7.

Show Gene links Show Anatomy links

Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor.

Fu C , Xiao Y , Zhou X , Sun Z .

???displayArticle.abstract???
The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09 Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R.

???displayArticle.pubmedLink??? 38965213
???displayArticle.pmcLink??? PMC11224282
???displayArticle.link??? Nat Commun
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: sult2a1 ube2k
GO keywords: endoplasmic reticulum signal peptide binding [+]

???attribute.lit??? ???displayArticles.show???

	Fig. 1: Structure of xlσ1R bound to a putative antagonist progesterone. a Structure of the xlσ1Runknown-lig homotrimer viewed perpendicular to the membrane from the β-barrel side. One protomer is colored in green and the other two in gray. The β-barrel lumen is rendered in orange surface throughout the manuscript. b Superposition of xlσ1Runknown-lig (green cartoon) and xlσ1Rprog-co (yellow cartoon). Five α helices (α1-α5) and the β-barrel (β1-β10) are labeled. The relative position of the ER membrane is indicated by a gray rectangle. c An unknown electron density (purple mesh) inside the β-barrel lumen (orange surface) of xlσ1Runknown-lig (green cartoon). The words “distal” and “proximal” describe the relative distances from different β-barrel lumen regions to the ER membrane. d A close-up view of the tadpole-shaped unknown density (purple mesh) from panel c. e A xlσ1Rprog-co protomer (yellow cartoon) containing a fish-shaped density (purple mesh) in the β-barrel lumen (orange surface). f Chemical structure diagram of progesterone. The diagram shows the α-face of progesterone’s steroid rings A to D. The backbone carbon atoms are numbered. g, h Fitting of progesterone (cyan sticks) into the fish-shaped density (purple mesh) of xlσ1Rprog-co (in yellow). i Hydrophobic interactions between bound progesterone (in cyan) and residues of xlσ1Rprog-co (yellow sticks). Distances from progesterone’s C20 carbonyl oxygen to nearby oxygen- or nitrogen-containing side chains are indicated by red dashed lines with distances labeled. E169 is shown as gray sticks. j MST fitting curves of progesterone binding to xlσ1R wild-type (WT, open circles) and E169A (open triangles). MST measurements were repeated three times (N = 3 biologically independent samples) with similar results. Data are presented as mean ± SD. Source data are provided as a Source Data file. In panels d, g, and h, water molecules are rendered as red spheres. In panels c, d, e, g, and h, the purple mesh shows the simulated annealing Fo-Fc (omit) map contoured at 3.0 σ level. In panels d and g, the blue mesh shows the simulated annealing 2Fo-Fc map contoured at 1.2 σ level. In panels c and e, only the β-barrel and α4/α5 of xlσ1R are shown for clearer views.
	Fig. 2: Indirect interaction between progesterone and xlσ1R mediated by water. a Electron densities (blue mesh) of six water molecules (red spheres) in the distal space of the β-barrel lumen (orange surface) of xlσ1Rprog-co (yellow cartoon). The blue mesh shows the simulated annealing 2Fo-Fc map contoured at 1.2 σ level for water molecules. Progesterone (cyan sticks) binds in the β-barrel lumen region proximal to the membrane. b A close-up view of panel a showing the distal space of the β-barrel lumen. Potential hydrogen bonds with the donor-acceptor distance of 2.7–3.6 Å are indicated by black dashed lines. Coordinating residues are shown as yellow sticks. Water403 is labeled. c Solvent accessibility analysis of xlσ1Rprog-co. The solvent-accessible space is displayed as red surface. The α4/α5-entrance and the hinge-entrance are indicated by black arrows. d The α4/α5-entrance surrounded by residues L179 (α4), L183 (α4), V200 (α5) and Y203 (α5) (yellow sticks). Viewed perpendicular to the membrane from the membrane side of xlσ1Rprog-co. e The hinge-entrance surrounded by residues F80 (β1), V81 (β1), N82 (β1), T186 (α4), and T195 (α5) (yellow sticks). f Two water molecules (red spheres) near the hinge-entrance in xlσ1Rprog-co. The shortest distance (~12 Å) between the hinge-entrance water and the distal β-barrel lumen water is indicated by a black dashed arrow. In panels a, c, d–f, only the β-barrel and α4/α5 of xlσ1R are shown for clearer views.
	Fig. 3: A side-entrance for potential water entry in xlσ1R. a Structure of xlσ1Rside-open with each asymmetric unit containing two trimers (six protomers). Protomers A and B are colored in green (side-closed conformation) and the rest in light blue (side-open conformation). b, c The side-closed conformation (b) and the side-open conformation (c). Residues 114-118 (β4/β5 loop) and 177-181 (α4) are displayed in surface mode. The location of the side-entrance is indicated by a black arrow. d Superposition of the side-open conformation (in light blue) onto the side-closed conformation (in green). Residues Y117 and W118 are displayed as sticks. The location of the side-entrance is indicated by a magenta dashed oval. e Solvent accessibility analysis of the side-open conformation viewed from the β-barrel side. The solvent-accessible space is shown as red surface, and residues capable of hydrogen bonding along the solvent path are shown as sticks. The side-entrance is indicated by a black arrow. In panels b, c, and e, only the β-barrel and α4/α5 of xlσ1R are shown for clearer views.
	Fig. 4: Structure of xlσ1R bound to a putative agonist DHEAS. a A xlσ1RDHEAS-I432 protomer (pink cartoon) containing an electron density (blue mesh) in the β-barrel lumen (orange surface). For a clearer view, only the β-barrel and α4/α5 of xlσ1R are shown. b Chemical structure diagram of DHEAS. The diagram shows the α-face of the steroid rings A to D of DHEAS. The backbone carbon atoms are numbered. c Density fitting of DHEAS (pink sticks) in the β-barrel lumen (orange surface) of xlσ1RDHEAS-I432 (pink cartoon). Water molecules in the distal β-barrel lumen are shown as red spheres. d Structural alignment of xlσ1Rprog-co (in yellow) and xlσ1RDHEAS-I432 (in pink). Progesterone, DHEAS and residues E169 and Y100 are shown as sticks. A potential hydrogen bond between the C3 sulfuric ester group of DHEAS and the E169 side chain is indicated by a pink dashed line with the donor-acceptor length labeled. Only α4/α5 of xlσ1R are shown for a clearer view. e The electron density (blue mesh) of DHEAS (pink sticks) in xlσ1RDHEAS-I432, showing two density protrusions towards the membrane. f MST fitting curves of DHEAS binding to xlσ1R WT (closed circles) and E169A (closed triangles). MST measurements were repeated three times (N = 3 biologically independent samples) with similar results. Data are presented as mean ± SD. Source data are provided as a Source Data file. g Electron densities (blue mesh) of two water molecules (red spheres) in the distal β-barrel lumen (orange surface) of xlσ1RDHEAS-I432. DHEAS and residue D123 are shown as sticks. Potential hydrogen bonds are indicated by black dashed lines. In panels a, c, e, and g, the blue mesh shows the simulated annealing 2Fo-Fc map contoured at 1.2 σ level.
	Fig. 5: A two-part-interaction model for ligand binding in σ1R. a A σ1R ligand (shown as surface) is usually elongated in shape and binds inside the β-barrel lumen of xlσ1R (yellow cartoon). Two parts of the ligand are colored in blue and brown, respectively. Residues that interact with the ligand are colored in green or gray. For a clearer view, only the β-barrel and α4/α5 of xlσ1R are shown. b A close-up view of panel a, showing details of xlσ1R-ligand interactions. Participating residues are shown as sticks. Green residues line the distal region of the β-barrel lumen (polar zone) and interact with the ligand’s polar moiety (or water; shown as blue surface). Gray residues line the proximal region of the β-barrel lumen (hydrophobic zone) and interact with the hydrophobic moiety of the ligand (shown as brown surface). Residue Y117 may participate in both polar and hydrophobic interactions, but is displayed as gray sticks in this panel. The side chain of E169 in xlσ1R (or E172 in hσ1R) is located near the junction between the polar and hydrophobic zones. c A generalized two-part-interaction model for σ1R ligands. The polar moiety (or water) and the hydrophobic moiety of the ligand are shown as a blue oval and a brown oval, respectively. The maximal lengths of the polar moiety and the hydrophobic moiety of a ligand are estimated to be ~8 Å and ~10 Å, respectively.
	Supplementary Figure 1. Electron density maps of xlσ1R structures generated in this study. (a) xlσ1Runknown-lig (PDB ID: 8W4D). (b) xlσ1Rprog-soak (PDB ID: 8W4C). (c) xlσ1Rprog-co (PDB ID: 8W4B). (d) xls1Rside-open (PDB ID: 8W4E). (e) xlσ1Rside-open-all (PDB ID: 8YBB). (f) xlσ1RDHEAS-I432 (PDB ID: 8WWB). (g)-(i) xlσ1RDHEAS-C2 (PDB ID: 8WUE) protomer A (Pose-1, panel g), protomer B (Pose-2, panel h) and protomer C (Pose-3, panel i). In all panels, xlσ1R structures are shown in light orange, and the blue mesh shows the simulated annealing 2Fo-Fc map contoured at 1.2 s level. In panels a, b, c and f, water molecules are shown as red spheres. In panels b, c, f, g, h and i, progesterone (panels b and c) or DHEAS (panels f, g, h and i) are shown as cyan sticks, and the purple mesh shows the simulated annealing Fo-Fc omit map contoured at 3.0 s level.
	Supplementary Figure 2. Structural features of xlσ1Runknown-lig and the xlσ1R-progesterone complex. (a) Crystal packing of xls1Runknown-lig in the I432 space group. Each asymmetric unit contains one xlσ1R protomer (green cartoon). The a1 helices of a xlσ1R trimer are shown as red cylinders. (b) Superposition of the xlσ1Rprog-co trimer (yellow cartoon) and the xls1Runknown-lig trimer (green cartoon). Viewed perpendicular to the membrane from the b-barrel side. (c) Superposition of xlσ1Runknown-lig (green cartoon) onto a previously reported xlσ1R structure (xlσ1Rclosed-endo, PDB entry 7W2B, blue cartoon). The relative position of the ER membrane is indicated by a grey rectangle. (d) Superposition of a xlσ1Rprog-soak protomer (orange tube) onto xlσ1Rprog-co (yellow tube). (e) Superposition of lumen-lining residues betweenxlσ1Runknown-lig (green sticks) and xls1Rprog-co (yellow sticks). (f) Superposition of xlσ1Rprog-co (in yellow) onto the hσ1R-haloperidol structure (in lime green, PDB entry 6DJZ). Progesterone, haloperidol and residue E172/E169 are shown as sticks. The b-barrel lumen is indicated by orange surface.
	Supplementary Figure 3. Sequence alignment of s1R homologs by ClustalW1,2. Alpha helices are indicated by cylinders and labeled from a1 to a5. The b-barrel is indicated by ten arrows labeled from b1 to b10. Residues participating in polar or hydrophobic interactions with ligands are highlighted in cyan or yellow, respectively. Residue Y117 is highlighted in both cyan and yellow to reflect that it may participate in both polar and hydrophobic interactions. The highly conserved residue E169 (xls1R)/E172 (hs1R) is underscored. Asterisks (*) indicate identical residues. Colons (:) indicate strong similarities. Periods (.) indicate weak similarities. xls1R, s1R from Xenopus laevis; ms1R, s1R from Mus musculus; hs1R, s1R from Homo sapiens.
	Supplementary Figure 4. Water near the hinge-entrance of xlσ1R. (a) Water molecules (yellow/green spheres) near the hinge-entrance of xlσ1Rprog-co (yellow cartoon) and xlσ1Runknownlig (green cartoon). (b) The shortest distance between the hinge-entrance water (red spheres) and the distal b-barrel lumen water (red spheres) in xlσ1Rprog-co is indicated by a black dashed line. Residues along the line are shown as sticks. In both panels, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views.
	Supplementary Figure 5. MD simulation of xlσ1Rprog-co. (a) Snapshot of a xlσ1Rprog-co monomer showing a closed side-entrance before MD simulation. (b)-(d) Snapshots of xlσ1Rprog-co showing an open side-entrance at 54th ns of MD run #1 (panel b), 17th ns of MD run #2 (panel c) and 98th ns of MD run #3 (panel d). The location of the side-entrance is indicated by a black arrow. Residues surrounding the side-entrance are displayed in surface mode. Among these residues, R116 (b4/b5 loop), S177 (a4) and F181 (a4) are colored in blue, red and yellow, respectively. In all panels, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views.
	Supplementary Figure 6. The side-open conformation of xlσ1R structures. (a) A trimer of the xlσ1Rside-open structure. Three protomers (A, B and C) are shown in different colors. Residues Y117 and W118 of one protomer and residues Q191 and F193 of the adjacent protomer are rendered in spheres. For a clearer view, a1 is not shown in this panel. (b) A close-up view of panel a, showing the contacting region between protomer A (side-closed, in green) and protomer C (side-open, in light blue). (c) A close-up view of panel a, showing the contacting region between protomer A (side-closed, in green) and protomer B (side-closed, in pink). (d) Structure of xlσ1Rside-open-all with each asymmetric unit containing two trimers (six protomers) in yellow orange. (e) Protomer A of xlσ1Rside-open-all. (f) Superposition of protomers A to F (all in yellow orange) of xlσ1Rside-open-all. In panels e and f, residues 114-118 (b4/b5 loop) and 177-181 (a4) are displayed in surface mode, and the location of the side-entrance is indicated by a black arrow. In panels e and f, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views
	Supplementary Figure 7. Comparison between xlσ1Rprog-co and xlσ1RDHEAS-I432. (a) Superposition of xls1RDHEAS-I432 (pink tube) onto xls1Rprog-co (yellow tube). (b) Superposition of lumen-lining residues between xlσ1RDHEAS-I432 (pink sticks) and xlσ1Rprog-co (yellow sticks). (c) Comparison of bound progesterone (yellow sticks) to DHEAS (pink sticks) in the b-barrel lumen (orange surface) of xlσ1Rprog-co (yellow cartoon) and xlσ1RDHEAS-I432 (pink cartoon), respectively.
	Supplementary Figure 8. Docking of potential steroid ligands into xlσ1R. (a)-(g) The top-scored docking models of progesterone (panel a), pregnenolone (panel b), pregnenolone sulfate (panel c), allopregnanolone (panel d), DHEA (panel e), DHEAS (panel f) and 16,17- didehydroprogesterone (panel g) using the protein portion of the xls1Rprog-co structure (see Methods). Docking models of all steroids are displayed as grey sticks. Progesterone and DHEAS from the xls1Rprog-co and xls1RDHEAS-I432 structures are shown as yellow (panel a) and pink sticks (panel f), respectively, and their binding locations are indicated by a yellow bar and a pink bar, respectively, on the right of each panel. (h) Published structures of hσ1R/ xlσ1R bound to synthetic ligands, which are shown as color sticks. Potential polar interactions between the basic nitrogen atom of ligands and the E169/E172 side chain of σ1R are indicated by black dashed lines. In all panels, only the b-barrel and a4/a5 of xlσ1R are shown for clearer views.
	Supplementary Figure 9. Structure of xlσ1RDHEAS-C2. (a) Six protomers in each asymmetric unit of xls1RDHEAS-C2 with three different DHEAS binding poses. DHEAS Pose-1 in protomers A and D (light orange cartoon); DHEAS Pose-2 in protomers B, E and F (cyan cartoon); DHEAS Pose-3 in protomer C (green cartoon). (b) Superposition of xlσ1RDHEAS-C2 Pose-2 (in cyan) onto xls1RDHEASI432 (in pink). DHEAS is shown as sticks. (c)-(e) Density fitting of DHEAS Pose-1 (light orange sticks, panel c), Pose-2 (cyan sticks, panel d), and Pose-3 (green sticks, panel e) in the b-barrel lumen (orange surface) of xlσ1RDHEAS-C2 protomer A (panel c), protomer B (panel d), and protomer C (panel e). In panels c, d and e, the blue mesh shows the simulated annealing 2Fo-Fc map contoured at 1.2 σ level.