Allosteric regulation of DegS protease subunits through a shared energy landscape

The PDZ domains of the trimeric DegS protease bind unassembled outer-membrane proteins (OMPs) that accumulate in the E. coli periplasm. This cooperative binding reaction triggers a proteolytic cascade that activates a transcriptional stress response. To dissect the mechanism of allosteric activation, we generated hybrid DegS trimers with different numbers of PDZ domains and/or protease-domain mutations. By studying the chemical reactivity and enzymatic properties of these hybrids, we show that all subunits experience a strongly coupled energetic landscape. For example, OMP-peptide binding to a single PDZ domain stimulates active-site chemical modification and proteolytic cleavage in the attached and neighboring protease domains. OMPpeptide binding relieves inhibitory PDZ interactions, whereas the interfaces between protease domains in the trimeric DegS core mediate positively cooperative activation driven both by substrate binding and inhibition relief. HtrA-family proteases consist of a protease domain, which employs a classical Ser-His-Asp catalytic triad and assembles into a stable trimer, and one or two PDZ domains, which regulate activity by binding the C-termini of regulatory or substrate molecules1. Human HtrA enzymes play roles in apoptosis, growth, and differentiation, and have been linked to arthritis, cancer, vascular disease, and macular and neural degeneration2–8, but much of our understanding of these proteases is based on studies of DegS, a bacterial ortholog. E. coli DegS is a single PDZ-domain protease that senses environmental stress in the periplasm by binding the C-termini of outer-membrane proteins (OMPs) that fail to assemble9. This binding event activates DegS proteolysis of a transmembrane anti-sigma factor, initiating an intramembrane proteolytic cascade that ultimately results in transcription of envelope-stress genes in the cytoplasm10. DegS trimers are anchored to the periplasmic face of the inner membrane, with each subunit containing a trypsin-like protease domain and a PDZ domain1,11. Heat shock and other stresses slow insertion of OMPs into the outer membrane, and unassembled OMPs accumulate in the periplasm, where their C-terminal Tyr-Xxx-Phe peptides bind to the DegS PDZ domains and activate cleavage of the RseA periplasmic domain (RseAP)9. RseA spans the inner membrane and its cytoplasmic domain binds and inhibits the σE transcription factor12–14. DegS cleavage of RseAP creates a new C-terminal residue that recruits RseP, an integral-membrane protease, resulting in a second cleavage of RseA and release of its cytoplasmic domain plus bound σE from the membrane15–18. AAA+ proteases then degrade the RseA portion of this complex19,20, liberating σE to activate transcription. Address correspondence to: [email protected]. Author contributions. R.V.M. performed all experiments. R.V.M. and R.T.S. contributed to experimental design, interpretation, and writing the manuscript. The authors declare no competing financial interests. NIH Public Access Author Manuscript Nat Chem Biol. Author manuscript; available in PMC 2013 August 01. Published in final edited form as: Nat Chem Biol. 2013 February ; 9(2): 90–96. doi:10.1038/nchembio.1135. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript The core of the DegS enzyme is formed by symmetric packing between its three protease domains, with peripheral PDZ domains contacting only the protease domain in the same subunit21,22 (Fig. 1a). The ligand-free PDZ domains inhibit proteolysis, as RseA is cleaved more than 100-fold faster by a DegSΔPDZ variant than by DegS in the absence of OMP peptides9,23,24. The site for OMP-peptide binding in each PDZ domain is ~25 Å from the nearest proteolytic active site in the trimer21, indicating that the mechanism of activation must be indirect. Many features of OMP activation of DegS cleavage of RseA can be explained by a Monod-Wyman-Changeux (MWC) model of allostery in which DegS trimers exist either in an inactive/tense conformation or in an active/relaxed conformation, with the binding of specific OMP peptides and/or the RseA substrate altering the equilibrium between these states24–27. Indeed, the trimeric protease domain displays two basic conformations, which correspond to active and inactive structures, in a large number of crystal structures21–24,27,28. The free energies and percentage of active enzymes for the different unbound or ligand-bound states of DegS and DegSΔPDZ (Fig. 1b) have been calculated based on MWC modeling of experimental data24,26. However, other models of DegS activation have been proposed in which OMP binding to a specific PDZ domain selectively tunes the cleavage activity of only one DegS subunit rather than the entire trimer21,28. Allostery is widely used to control biological reactions, and the molecular mechanisms that mediate and control the underlying cooperative conformational changes are therefore of substantial interest.29,30 To test the mechanism of allosteric activation by OMP peptides, here we generate and study the enzymatic properties of asymmetric DegS trimers that lack one or two PDZ domains. By using active-site chemical profiling31 and/or placing mutations that inactivate specific protease domains in these trimers, we show that OMP-peptide binding to a single PDZ domain affects the activity of the protease domain in the same subunit and neighboring subunits. Similarly, mutations that stabilize or destabilize the active conformation of one protease domain affect the activities of neighboring protease domains. Our results support a model in which the allosteric conformations and thus the proteolytic activities of each DegS subunit are cooperatively coupled through a shared free-energy landscape of the trimeric core. OMP-peptide binding relieves inhibitory PDZ interactions, allowing positively cooperative proteolytic activation driven in part by the intrinsic energetic landscape of the trimeric core and in part by substrate binding.