Expression, crystallization and crystallographic analysis of DegS, a stress sensor of the bacterial periplasm.

Regulated proteolysis is a key event in transmembrane signalling between intercellular compartments. In Escherichia coli, a protein DegS has been identified as being a periplasmic stress sensor for unfolded or misfolded outer membrane proteins (OMPs). Activation of DegS initiates a proteolytic cascade which results in the transcription of periplasmic genes under sigmaE control, most importantly chaperones and proteases. DegS has been cloned and expressed as full-length protein and in an N-terminally truncated form. Both proteins were tested for crystallization and two forms of well diffracting crystals of the truncated form were obtained. Crystals of form I diffract to 3.5 A and belong to space group P2(1)3, while crystals of form II diffract to 2.2 A and belong to space group I23. Crystals of form II were soaked with a consensus peptide representing the C-termini of outer membrane proteins and data to 2.4 A resolution were collected. Molecular-replacement trials using a homologous protease domain indicate the presence of two molecules in the asymmetric unit of crystal form I. The correctness of the molecular-replacement solution was verified by identifying radiation-damage-induced structural changes.