Fig. 4: Conformational and dynamic changes in SpEgtUC upon ET binding.
From: Discovery and structure of a widespread bacterial ABC transporter specific for ergothioneine
a AlphaFold2 model of “open” apo SpEgtUC (gray D2, green D1, magenta linkers). F277W and the L374C site of bimane labeling are shown as an open hexagon and a cyan circle, respectively. b The “closed” crystal structure of ET-bound SBD (gray D2, blue D1, red linkers), with F277W and L374C shown as in a. c Bimane fluorescence emission spectra of bimane-labeled L374C/F277W SpEgtUC with (red) and without (black) ET. d Normalized change in fluorescence emission at 480 nm upon titration of bimane-labeled L374C/F277W SpEgtUC with ET in a position-induced fluorescence quenching (PiFQ) experiment51. Each titration point is shown as the mean and standard deviation of two independent replicates. See Table 1 for fitted parameters. e 1H,15N TROSY spectrum of apo EgtUC (residue-specific assignments in Supplementary Fig. 12). f 1H,15N TROSY spectrum of EgtUC bound to equimolar ET (Supplementary Fig. 13). g Backbone chemical shift perturbations (CSPs) upon binding ET for each residue in SpEgtUC. Assignments are missing for residues H310 and V485 in the ET-bound state (shaded pink). Prolines are shaded gray. h CSPs of ET binding painted onto the crystal structure of ET-bound SpEgtUC, with large chemical shift changes shown as thick, red tubes. i B-factors plotted on the crystal structure of SpEgtUCCTT, with high values shown as thick, red tubes, revealing low B-factors in the interdomain linkers, and comparable to the B-factors of high occupancy solvent molecules. j Overlay of 1H,15N TROSY spectra of apo and ET-bound EgtUC, zoomed to the region where the arginine side chain peaks are folded into this spectral window. k H-bond networks of arginine side chains (R320, R379, R404) found in the crystal structure. l 1H,15N heteronuclear NOEs for these three arginine side chains. Heteronuclear NOE data were recorded as one replicate, with error bars indicating the uncertainty derived from spectral noise. Source data for those data shown in panel d are provided as a Source Data file.
