Figure 1.

DegS and hybrid trimers containing mixtures of full length (S) and ΔPDZ (Δ) subunits. (a) Each subunit of the DegS trimer contains a trypsin-like protease domain (darker color; active site shown in red) and a peripheral PDZ domain (lighter color). Contacts between protease domains stabilize the trimer. OMP peptides (CPK representation) bind to the PDZ domains. The structure shown (3gcn)²⁷ is the proteolytically active conformation. (b) Free-energy differences for DegS and DegS^ΔPDZ with and without saturating RseA^P substrate and/or OMP peptide (Tyr-Tyr-Phe) were calculated (ΔG = − RT ln ([active]/[inactive]) from values taken from references 24 and 26. The right axis shows the percentage of active trimers for each molecular species. DegS^ΔPDZ has no binding site for OMP peptide. (c) The denaturation-renaturation protocol used to generate hybrid trimers is shown in the upper portion of the panel. The lower portion shows separation of hybrid trimers by ion-exchange chromatography. The inset shows SDS-PAGE of the four major peaks (see Supplementary Fig. 2 for complete gel). (d) The steady-state rate of RseA^P (200 μM) cleavage by the SSΔ or SΔΔ variants showed a linear dependence (R² > 0.98) on enzyme concentration. Reactions contained 150 μM Tyr-Tyr-Phe tripeptide.