Crystal structure of DegP from Escherichia coli

DegP is a heat-shock protein which is localized in the periplasm of Escherichia coli. It is a common protein quality control factor and represents the only protein that can alternate between the two antagonistic activities of a protease and a chaperone in a temperature-dependent manner. In this study the crystal structure of the hexameric form of DegPs2ioA from Escherichia coli was solved at 2.8A resolution by multiple anomalous dispersion phasing. As the protein was crystallized at room temperature, this structure represents the chaperone conformation. Each DegP monomer is built up by a trypsin-like serine protease domain and two consecutive PDZ domains. The hexamer is a dimer of trimers with crystallographic D3 symmetry. Oligomerization is mediated by the protease domains and results in the formation of an internal cavity where the active sites are located. Access towards the internal cavity is controlled by the flexible PDZ domains. The protease activity is absent because access towards the active sites is blocked by the interaction of several surface loops. Furthermore, the active site geometry is distorted. The crystal structure of the wild-type DegP confirmed that the inactive conformation is not due to the artificial serine to alanine mutation of the DegPs2ioA structure but represents an inherent feature of the chaperone state to avoid unwanted proteolysis at room temperature. The crystal structure of DegP in complex with the covalent serine protease inhibitor diisopropyl fluorophosphate could not preserve the protease conformation. Analysis of degradation products by mass spectrometry revealed that the product length varies between 6 and 25 amino acid residues with a clear preference for small hydrophobic residues in the PI position. Furthermore, time-dependent analyses of degradation products by high-performance liquid chromatography showed that DegP degrades its substrates in a processive fashion, similar to other cage-forming proteases