HtrA protease-chaperones

HtrA - as housekeeping PDZ-protease

The high temperature requirement A (HtrA) proteins are ubiquitously expressed PDZ-proteases that are critical to maintain protein homeostasis in extracytoplasmic compartments. In contrast to other quality control factors, HtrA proteases are capable to function as chaperone stabilizing specific client proteins. As HtrA proteins combine the dual activities of a protease and a chaperone on a single polypeptide, they represent a unique model system to monitor the partitioning of damaged proteins between digestive and refolding pathways. With regards to their structures, HtrA proteins associate via their protease domains to form trimers, the building block of all HtrA oligomers. The projecting PDZ domains either participate in protein degradation presenting substrates to the protease or mediate the binding of allosteric activators that stimulate protease function. HtrA housekeeping proteases are implicated in important cellular processes such as bacterial virulence, photosynthesis, organization of the extracellular matrix, cell proliferation and ageing. Consistently, the loss of mammalian HtrA activity is connected with severe diseases, including arthritis, cancer, familial ischemic cerebral small-vessel disease and age-related macular degeneration, as well as Parkinson's disease and Alzheimer's disease. Our work aims to better understand the molecular basis of these HtrA-connected pathologies.

Our findings: 2008 Mechanism of a protease-chaperone * 2010 Architecture of the HtrA ancestor DegQ * 2011 Substrate-induced activation of human HtrA1



2008 - Structural basis of the DegP protease-chaperone

A combination of chaperone and protease function in a single protein could provide a direct and rapid response to protein folding problems. The heat-shock protein DegP combines these dual activities in a single polypeptide chain and thus offers unique possibilities to investigate how cells distinguish between proteins that can be refolded and “hopeless” cases that need to be degraded. DegP from E. coli is a central protease of the protein-quality-control system in the bacterial envelope and, in parallel, contributes to OMP biogenesis ensuring the safe transit of OMP precursors to the outer membrane. To investigate the molecular basis of these dual activities, we characterized different DegP/substrate complexes. Binding of misfolded proteins transformed the resting DegP hexamer into large, catalytically active 12- and 24-meric multimers. Structural analysis of these particles revealed that DegP assembles a huge protein packaging device, whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity serves antagonistic functions. While encapsulation of folded OMP protomers is protective permitting the safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber.


2010 - Organization of the housekeeping protease-chaperone DegQ

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of E. coli is monitored under non-stress conditions by the HtrA protease DegQ. In this work we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1-L3-LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope.


2011 - Substrate-induced activation of human HtrA1

Mammalian HtrA1 contains an N-terminal domain of unknown function, a serine protease domain and one C-terminal PDZ domain. Despite its physiological importance in quality-controlling the extracellular matrix, little is known about the molecular mechanism of HtrA1. We therefore crystallized human HtrA1 in its active and inactive conformations and performed biochemical analyses to address its mechanistic features. Although the structure of HtrA1 is similar to that of other HtrA proteases, there are striking mechanistic differences. In bacterial HtrAs, PDZ domains act as sensors of protein folding stress, substrate-binding sites and regulators of the enzymatic activity and are required for oligomerization. Unexpectedly, in human HtrA1 the PDZ domain is largely dispensable for protease function. Indeed, our data indicate that ligand binding to the protease domain is sufficient to rearrange the activation domain and stimulate protease activity, as the substrate itself completes and thereby remodels the activation domain. Accordingly, the activity of HtrA1 is controlled by induced-fit substrate binding and does not depend on allosteric ligands acting in trans such as those seen in DegP or DegS. This auto-activation provides an unprecedented mechanistic facet of HtrA protease regulation and implies that HtrA1 is subject to further regulatory mechanisms that might control, for example, its specific cellular localization.