Structures of oxysterol sensor EBI2/GPR183, a key regulator of the immune response

Abstract

Oxysterols induce the migration of B-lymphocytes and dendritic cells to interfollicular regions of lymphoid tissues through binding the EBI2 (GPR183) to stimulate effective adaptive immunity and antibody production during infection. Aberrant EBI2 signaling is implicated in inflammatory bowel disease, sclerosis, and infectious disease. Here, we report the cryo-EM structures of an EBI2-G_i signaling complex with its endogenous agonist 7α,25-OHC and that of an inactive EBI2 bound to the inverse agonist GSK682753A. These structures reveal an agonist binding site for the oxysterol and a potential ligand entrance site exposed to the lipid bilayer. Mutations within the oxysterol binding site and the Gα_i interface attenuate G protein signaling and abolish oxysterol-mediated cell migration indicating that G protein signaling directly involves in the oxysterol-EBI2 pathway. Together, these findings provide new insight into how EBI2 is activated by an oxysterol ligand and will facilitate the development of therapeutic approaches that target EBI2-linked diseases.

Keywords: EBI2; GPCR; GPR183; cryo-EM; immunity; immunocyte migration; oxysterol; signaling.

Figure 1.. Functional characterization of human EBI2 and its structures

(A) Concentration curve of oxysterol-mediated G protein activation. The EC₅₀ of 7α,25-OHC (structure shown) is 60 nM. Data are mean ± s.d. (n=3). Chemical structures of 27-OHC and 7α,25-OHC are shown on the top. C-7, C-25, and C-27 positions of oxysterols are indicated. (B) Inverse agonists inhibit the activity of EBI2. The inverse agonist at various concentrations was supplemented into the protein in the presence of 100 nM 7α,25-OHC. The IC₅₀ of GSK682753A is 0.35 μM and the IC₅₀ of NIBR189 is 0.23 μM. Data are mean ± s.d. (n=3). Chemical structures of NIBR189 and GSK682753A are shown on the top. (C) Cryo-EM structure of EBI2-BRIL-Fab-Nb complex. One class of the 2D classification from cryoSPARC is shown. GSK682753A is shown in cyan sticks. (D) Overall structure of EBI2-G_i complex. The 7α,25-OHC is shown in yellow sticks. Each structural element is indicated. The maps of GSK682753A and 7α,25-OHC are shown in mesh at 5σ level. See also Figure S1, S2, and Table S1, S2.

Figure 2.. Structure of GSK682753A bound EBI2 in the inactive state

(A) Overall structure viewed from membrane side. (B) Extracellular view of EBI2. The disulfide bond is shown in yellow sticks. Each structural element is indicated. (C) Electrostatic surface representation of EBI2 showing the inverse agonist binding pocket. The interaction details in the dashed frame are shown in (D). (D) The position of the GSK682753A in EBI2. The putative GSK682753A is shown in cyan sticks. Residues are represented as sticks. See also Figure S1 and S3.

Figure 3.. Structure of 7α,25-OHC bound EBI2 in the active state

(A) Overall structure viewed from membrane side. The 7α,25-OHC is shown in yellow sticks. (B) The extracellular view of EBI2. The potential entrance of oxysterol is indicated. (C) The interaction details between 7α,25-OHC and EBI2. (D) Chemical structures of 7α,25-OHC, 7β,25-OHC, and 7α,27-OHC. C-7, C-25, and C-27 positions of dihydroxycholesterols are indicated. (E) Electrostatic surface representation of EBI2 showing the putative sterol entrance. (F) Structures of sterol derivative-bound GPBAR1 and SMO in the active states. The 3-hydroxyl group of the sterol derivative is indicated by arrows and the putative sterol entrance of SMO (between G416 and A459) is indicated. PDB codes are shown in parenthesis. Residues are represented as sticks. (G) Activities of A161F, P165F and A200L mutants relative to wild-type EBI2 measured by fluorescence-based GTP turnover assay. Data are mean ± s.d. (n=3). *** p<0.001,****p<0.0001, unpaired Student’s t test. See also Figure S2 and S4.

Figure 4.. Comparison of active EBI2 with its inactive state

(A) Structural comparison between 7α,25-OHC-bound EBI2 (dark blue) with its inactive state (light blue) viewed from the side of the membrane. The comparison of TM6 in both the states is shown. (B) Extracellular (upper panel) and cytosolic (lower panel) views. The movements of TMs after activation are indicated by red arrows. (C and D) The potential mechanism of 7α,25-OHC-mediated EBI2 activation. (E) Activities of EBI2 mutants relative to wild-type EBI2 measured by fluorescence-based GTP turnover assay. Data are mean ± s.d. (n=3). **p<0.01; ****p<0.0001, unpaired Student’s t-test. (F) Interaction details between EBI2 and Gα_i. The residues involved in ligand binding and conformational changes are shown in sticks. The hydrophilic interactions are indicated by dashed lines. See also Figure S5 and S6.

Figure 5.. Functional analysis of key residues of EBI2

(A) The fluorescence-based GTP turnover assays. The protein was mixed with heterotrimeric G_i in presence of 100 nM 7α,25-OHC for the measurement. Data are mean ± s.d. (n=4). (B) U937 cell migration assays. The medium contained 10 nM 7α,25-OHC and cells were measured using CCK-8 dye for cell counting at 450 nm. Data are mean ± s.d. (n=4). (C) Localization of EBI2 variants in cells. The HEK293 cells expressing EBI2-GFP were stained with Hoechst (blue). Scale bar, 10 µm. Fluorescence microscopy was performed as described in Methods. See also Figure S7.