Fig. 5: Contacts between ClpP β-hairpins and ClpC AAA-2 domains affect degradation activity.

a Topological scheme of the ClpC AAA-2 domain involved in contacts with the ClpP β-hairpins. To facilitate the visualization, each region involved in contacts is indicated and colored. b Ribbon model of the same regions labeled and colored according to (a). c, d Disulfide crosslinking experiments of neighboring residues of the ClpP β-hairpin and ClpC residues of the extensive P-loop (c) and allo-R region (d). ClpC wild type (WT), ClpC-D667C, ClpP wild type (WT), ClpC-T11C and MecA were incubated in the presence of 2 mM ATPγS as indicated. Disulfide crosslinking was induced under oxidizing conditions (+Cu(Phe3)). Presence of β-mercaptoethanol (β-MeOH) in the SDS sample buffer is indicated (*). Crosslink products were analyzed by SDS-PAGE. Positions of ClpP-ClpP and ClpC-ClpP disulfide crosslinks are indicated. e Subunit by subunit, visualization of the contacts between ClpP β-hairpins residues and ClpC residues of the allo-W, allo-R and P-loops regions. The sequence of contacts from ClpC subunit a to subunit f around the ClpC AAA-2 ring shows a shift of contacts from one tip to the other of the long allo-R helix. f LY-AMC degradation was monitored in the presence of ClpP wild type (WT) and indicated mutants in the absence or presence of ADEP1. Degradation rates were determined and set to 100% for ClpP WT (- ADEP1). g FITC-casein degradation rates by ClpP/ADEP1 or MecA/ClpC/ClpP (WT or mutants) were determined. Degradation activities of ClpP WT reactions were set to 100%. h Degradation rates of GFP-SsrA by MecA/ClpC/ClpP (WT or mutants) were determined. Degradation activity of ClpP WT was set to 100%. Error bars represent standard deviations (n = 3) (f–h).