Fig. 1: Cysteine trapping of Cys²-Sec, Cys⁵-Sec, Cys⁶-Sec, Cys⁷-Sec to cysteine mutants of non-dimerizing SecR(G264A,I268A) construct expressed on intact cells.

Shown in (A) are representative autoradiographs of 10% SDS-PAGE gels used to separate products of cysteine trapping of SecR mutants across ECL2 and ECL3 expressed in COS-1 cells by each noted probe. Gels were run in the absence of any reducing agent, and control receptor labeling on each gel was detected using key cysteine mutants of WT SecR. Shown also are the densitometric analysis of data from three to five independent experiments (Cys²-Sec, n = 4; Cys⁵-Sec and Cys⁶-Sec, n = 5; and Cys⁷-Sec, n = 3), with dots illustrating each data point. The receptor labeling signal was calculated relative to the intensity of the residue with the highest labeling using that probe across all regions. Sites of significant labeling above background were determined using one-way ANOVA with Dunnett’s post-test, with P < 0.001 considered to be significant (absolute values shown in Supplementary Table 3). In (A), these are colored red and marked with *. B provides a schematic representation of the labeling across all regions, showing the WT SecR residue(s) with the highest labeling intensity previously published^11,20,21 in blue, and the significantly labeled residues identified in the current work with the non-dimerizing mutant SecR(G264A,I268A) shown in red. C schematically illustrates these sites of cysteine trapping of the non-dimerizing mutant SecR (red spheres) along with the highest sites of covalent labeling of WT SecR previously reported¹¹ (blue spheres), as mapped onto our published cryo-EM structure of monomeric SecR in complex with human secretin and G protein¹¹ (SecR models displayed in ChimeraX version 1.6.1).