Figure 1: ADEP cooperatively binds and stabilizes SaClpP.
From: AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control
(a) Binding of ADEP to the hydrophobic pocket on the apical surface of ClpP causes a conformational change that opens the axial substrate entry pore of ClpP (PDB IDs: 1TYF, 3MT6). (b) Structure of ADEP7. (c) ADEP7-induced degradation of FITC–casein by SaClpP (0.5 μM) indicates positive cooperativity (Hill coefficient h=2.0±0.1; EC50=3.1±0.1 μM). (d) ITC titration of ADEP7 (500 μM) into a solution of SaClpP (50 μM) shows positive cooperative binding, which prevents standard data analysis. (e) ITC titration of SaClpP (617 μM) into a solution of ADEP7 (62 μM). Parameters obtained from this experiment are: stoichiometry factor n=1.01±0.02; Kd=2.1±0.5 μM; ΔH=−3369±100 cal mol−1; ΔS=13.8 cal mol−1 K−1. The stoichiometry factor refers to the ratio of monomeric SaClpP and ADEP7. A value of 1 is equivalent to 14 ADEP molecules binding per SaClpP14. (f) Thermal shift assays reveal strong stabilization of SaClpP folding by ADEP7 in a concentration-dependent manner (melting temperature of free SaClpP: 58.3 °C; ADEP7 concentration in upper panel: 12 μM; EC50=2.7±0.3 μM). Plotted data in c and f are mean±s.d. (N=3). Parameters determined from curve fits in c,e and f are given with fitting errors.
