Structure-function analysis of DegP

Proteases and chaperones together serve to maintain quality control of cellular proteins. The 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 (HtrA) exhibits these dual functionalities 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 is a widely conserved heat-shock protein found in most organisms. The prokaryotic proteins have been attributed to the tolerance against various folding stresses as well as to pathogenicity. Human homologues are believed to be involved in arthritis, cell growth, unfolded protein response, programmed cell death (apoptosis) and ageing. Although the structure of the DegP chaperone has been determined, several questions remained open that are related to the structure of the protease form. For example, what is the molecular basis for the switch in activity and how can this be triggered by a change in temperature? The active protease itself will also be of great interest in order to identify similarities and differences with related serine proteases. As a starting point we will use different DegP constructs to screen for potent inhibitors, which should be instrumental to stabilize and crystallize the protease form. Furthermore we plan to extend the structure-function analysis of E. coli DegP to eukaryotic and "pathogenic" homologs and to functionally related PDZ-proteases like YaeL, DegQ and Tsp. Although the protease domains of these proteins belong to different classes, they share a homologous PDZ domain that appears to be crucial for regulating activity. Our project aims to investigate this novel regulatory mechanims and to obtain insight how cellular factors channel misfolded proteins into either digestive or refolding pathways.

All living organisms employ dedicated chaperones and proteases to monitor and control the state of cellular proteins. Failure of this quality control can lead to protein aggregation, a malfunction correlated with fatal protein folding diseases. The protease-chaperone DegP, the central housekeeping protein in the bacterial cell envelope, represents a unique model system for uncovering mechanisms that protect cells from aberrant proteins as it combines digestive and remodelling activities on a single polypeptide. To investigate the molecular basis of the dual activities of DegP we characterized different complexes with substrate. Our data show that DegP represents another high-symmetry packaging device, whose central compartment is used to sequester unfolded proteins and to partition them between refolding and degradation pathways. In a first step, DegP has to sort out aberrant proteins that exhibit partially folded, aggregation-prone structures from properly folded proteins. The hexamer of DegP, DegP6 , appears to function as a substrate filter, as only unfolded proteins are capable of entering the cavity and assembling the functional protease-chaperone. Binding of misfolded proteins transforms the `resting` DegP6 into large, catalytically active 12- and 24-meric multimers, DegP12/24. Moreover, structural and biochemical analysis of these particles revealed that the central compartment of DegP is adaptable to the size and concentration of substrate and that the inner cavity serves antagonistic functions. While encapsulation of folded outer membrane proteins is protective and might allow safe transit through the periplasm, irreversibly damaged proteins are eliminated in the molecular reaction chamber. Oligomer re-assembly and concomitant activation upon substrate binding may also be critical in regulating related human DegP proteases implicated in protein folding diseases. In contrast to the ATP-dependent cytosolic proteases and chaperones, the molecular mechanisms of the extra- cytosolic factors are largely unknown. To address this question, we performed a comprehensive analysis of the protease function of DegP. Our data reveal an unprecedented mechanism of how DegP processively degrades misfolded proteins into peptides of defined size by employing a molecular ruler comprised of the PDZ domain, a well characterized protein-protein interaction module, and the proteolytic site. Furthermore, allosteric activation by peptides earmarking unstable proteins ensures the regulated and rapid elimination of misfolded proteins upon folding stress. Compared to the cytosolic proteases, the regulatory features of DegP are established by entirely different mechanisms reflecting the convergent evolution of an extra-cytosolic housekeeping protease.