Fig. 3: The PL-domain of PlpD associates with the POTRA domain in vivo.
a AlphaFold model of PlpD monomer (UniParc: UPI00054992B6) color-coded as in Fig. 1a. A portion of the POTRA and PL-domains is enlarged in the box on the right to highlight the relative location and orientation of the selected residues that were mutated to cysteine [PL-domain residues Q135 (blue), N136 (purple), L145 (cyan) and V146 (orange), and POTRA domain residues Q383 (light green) and V388 (dark green)]. b SEH88 transformed with pSEH171, pSEH172, pSEH202, pSEH203, pSEH211, pSEH238, pSEH264 or pSEH266 were grown and the expression of the indicated plpD cysteine mutants was induced as described in Fig. 1b. Aliquots were then either mock-treated or treated with the oxidant 4-DPS and samples were analyzed by immunoblot using the anti-PlpDC antiserum. Both the native uncrosslinked PlpD protein ( ~ 82 kDa) and an intra-molecularly crosslinked form ( ~ 100 kDa) were detected. The ~100 kDa bands were confirmed to result from disulfide bond formation by adding DTT to the samples prior to SDS-PAGE (see Fig. S8). The colors of the residue labels correspond to those in (a). c Quantitation of intra-molecular disulfide bond formation between cysteine residues located in the PL-domain and POTRA domain. The levels of disulfide bond formation in mock-treated cells (‘spontaneous’, Ox-) and 4-DPS-treated cells (‘catalyzed’, Ox + ) are shown. Bars = median, N = 3 biologically independent samples that each contained cells grown from a different colony. One-sided ANOVA and multiple comparison tests are shown in Table S4.
