Fig. 4: Blocking the putative entrance in PATH2.
From: An open-like conformation of the sigma-1 receptor reveals its ligand entry pathway
a One protomer of xlσ1Ropen-endo viewed perpendicular to the membrane from the membrane side. Left, the residues surrounding the entrance are displayed in sticks. Right, the surface representation shows an “egg” shape of the entrance residues in three parts: the rounded end (in blue), the middle (in yellow), and the pointed end (in red). b, c The stoichiometry (available binding site number per protomer, panel b) and the equilibrium dissociation constant (Kd, panel c) determined by ITC for the xlσ1R-C179/C203 protein in the indicated conditions. All ITC measurements were repeated with biologically independent samples. “Before oxidation”: n = 5. “After oxidation”: n = 3. “Re-reduction”: n = 4. Two-tailed Student’s t-test was performed between the conditions of “Before oxidation” and “After oxidation”, and between the conditions of “After oxidation” and “Re-reduction”. The p values are provided in a Source Data file. **p < 0.01. d Modification of mPEG-Mal-5K to the xlσ1R-C179/C203 protein in the indicated conditions analyzed by SDS-PAGE and Coomassie blue staining. The wild-type (WT) xlσ1R served as the control. The pound sign (#) indicates that the protein contains the extra C91S mutation (see “Methods” for details). The black arrowheads labeled with 0, +1, and +2 indicate the band positions without, with one, and with two mPEG-Mal-5K modification(s). The red arrowhead indicates a minor impurity band near 50 kDa, which does not affect the gel analysis. The modification experiment was repeated three times independently with similar results. e, f The stoichiometry (available binding site number per protomer, panel e) and the equilibrium dissociation constant (Kd, panel f) determined by ITC for the indicated xlσ1R proteins before and after the mPEG-Mal-5K modification. All ITC measurements were repeated with biologically independent samples. “Before modification”: n = 4 for xlσ1R WT, n = 4 for xlσ1R-C179, n = 3 for xlσ1R-C203. “After modification”: n = 6 for xlσ1R WT, n = 4 for xlσ1R-C179, n = 4 for xlσ1R-C203. Two-tailed Student’s t-test was performed between the groups of “Before modification” and “After modification”. The p values are provided in a Source Data file. **p < 0.01. For panels b, c, e, f, data are presented as mean + SD in the bar graphs, which are overlaid with corresponding data points shown as white dots. Source data are provided as a Source Data file.
