Figure 3: Binding of ADEP to ClpP induces a conformational switch to the active, extended ClpP barrel conformation.

(a) Cartoon representation of a ClpP monomer showing the position of the ADEP binding site, the catalytic triad (S98, H123, D172) and the oligomerization sensor R171 (PDB ID: 3MT6). (b) Analytical size exclusion chromatograms of wild-type and mutant SaClpP. (c) Analytical ultracentrifugation results for selected mutants. Sedimentation coefficients were obtained as the centres of Gauss functions whose fitting errors are given. (d) FITC–casein degradation activity of wild-type and mutant SaClpP (1 μM) in the presence or absence of ADEP7 (12 μM). Mean±s.d. of initial slopes are given. (e) Peptidase activity of wild-type and mutant SaClpP (0.5 μM) in the presence or absence of ADEP7 (12 μM). Mean±s.d. of initial slopes are given. (f) Inter-atomic pair distance distribution functions (PDDF) of tag-free SaClpP proteins treated with either DMSO or ADEP4 (1:1 mixture, 380 μM, 0.6% DMSO). (g) PDDF of tag-free SaClpP with/without ADEP7 (1:1 mixture, 380 μM, 0.6% DMSO). (h) Comparison of measured (ClpP wt) and predicted (3QWD, 4EMM, 3V5E) inter-atomic pair distance distribution functions. (i) Ab initio low-resolution models of SAXS scattering curves from SaClpP-D172N superimposed with SaClpP in the compressed conformation (left), as well as from SaClpP-D172N+ADEP4 superimposed with SaClpP in the extended conformation (right). (j) Dynamic light scattering results of wild type and D172N SaClpP treated with either DMSO or ADEP7 (1:1 mixture, 310 μM, 3% DMSO). Please note the axis break. Mean±s.e.m.; **P<0.01, ***P<0.001 (independent two-sample t-tests, N=10, with each of these 10 results established from 10 technical replicates, equal variances not assumed). UV, ultraviolet.