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. Author manuscript; available in PMC: 2016 Jul 2.
Published in final edited form as: Mol Cell. 2015 Jun 11;59(1):104–116. doi: 10.1016/j.molcel.2015.05.014

Figure 4. Phosphorylation modulates CpdR function through its response regulator output face.

Figure 4

(A) Effect of phosphorylation by the cognate CckA/ChpT phosphorelay on wildtype CpdR (WT) or the non-phosphorylatable variant CpdRD51A in mediating PdeA delivery to ClpXP. CpdR WT and CpdRD51A proteins were preincubated with the phosphorelay. PdeA detected by Western using α-PdeA due to overlapping bands. (B) SEC profiles of CpdR WT/CpdRD51A (±phosphorelay/ATP/ClpX). (C) SEC profiles of PdeA (detected by Western using α-PdeA) in the presence of CpdR WT/D51A (preincubated with phosphorelay/ATP) and ClpX/ATPγS. (D) Phyre homology model (RcsC receiver domain as template) for CpdR. Predicted α4-β5-α5 signaling output face (yellow), with conserved residues amongst α-proteobacteria CpdR orthologs (orange). (E) Alanine variants of CpdR H104 and R106 of CpdR were examined for their ability to mediate ClpXP degradation of GFP-PdeA. (F) CpdR variants characterized by fitting the initial rates of degradation of GFP-PdeA as a function of increasing adaptor concentration (Kactivation and Vmax reported; see Methods), data plotted as mean ± SD, n = 4. (G) SEC profiles of the CpdR variants (±ClpX). (H) Two-hybrid interactions between CpdR variants and ClpX/PdeA using E. coli Δcya reporter strains. Representative images on McConkey agar, and beta-galactosidase (β-gal) activity from 4 independent colonies (raw values, mean ± SD) are shown. See also Figure S4.