Clausen Group Research

Molecular mechanisms of protein quality control

The misfolding and aggregation of protein molecules is a major threat to all living organisms. Cells have therefore evolved a sophisticated network of molecular chaperones and proteases to prevent protein aggregation (Fig. 1A). In addition, protein quality control and regulatory proteolysis are important mechanisms in the defense line of several bacterial pathogens. My group is performing a structure-function analysis of prokaryotic and eukaryotic factors that combat folding stress and, in parallel, ensure controlled digestion of specific target proteins. A better understanding of protein quality control might disclose novel strategies to counteract protein folding diseases and bacterial pathogenicity.

Housekeeping HtrA proteases - Guards of the extracytosolic compartment

Figure 1 (Click to view legend)

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 (HtrA) can switch between these dual functionalities in a temperature-dependent manner (Fig. 1B), and thus offers unique possibilities to investigate how cells distinguish between proteins that can be refolded and “hopeless” cases that need to be degraded.

Figure 2 (Click to view legend)

DegP from E. coli is a central component of the protein-quality-control system in the bacterial envelope that is involved in eliminating misfolded proteins and in OMP (outer membrane protein) biogenesis. 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 (Fig. 2), 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 and might permit safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. Oligomer re-assembly and concomitant activation upon substrate binding may also be critical in regulating other HtrA proteases that promote diverse biological functions. Our current data provide excellent leads to address the molecular mechanisms of human DegP homologues, homologues from pathogenic bacteria, and functionally related PDZ proteases that exert housekeeping functions in mitochondria, chloroplasts and the extracellular space. Moreover, the identified activation mechanism should be helpful to obtain structural data concerning protease-substrate or protease-inhibitor complexes, which are indispensable for understanding how aberrant proteins are partitioned between refolding and degradation pathways.

The CtsR/McsB stress response

Figure 3 (Click to view legend)

All cells have evolved highly efficient signaling pathways that sense the presence of damaged proteins and transmit the signal “folding stress” to dedicated transcription factors, which then adjust the expression of the protein-quality-control factors. One of the most intensely studied stress-response pathways is the bacterial heat-shock system. In B. subtilis, the transcription factor CtsR is a major component of this system that represses the clpC heat shock operon, the clpE and clpP genes, by binding specifically to a 7-nucleotide direct repeat sequence located upstream of the transcriptional start sites (Fig.3A). Stress-induced transcription of the clp genes depends on the inactivation of CtsR by McsB. To delineate the molecular basis of CtsR and McsB in the bacterial stress response, we screened the respective proteins from various Gram-positive bacteria for recombinant production, and succeeded in reconstituting the Bacillus stearothermophilus CtsR/McsB system in vitro.

The crystal structure of the CtsR repressor in complex with DNA revealed how partial asymmetry in a dimeric transcription factor allows high affinity binding to tandem DNA repeats. Moreover, biochemical characterization of McsB highlighted a novel protein kinase activity. McsB specifically phosphorylates arginine residues in the DNA-binding domain of CtsR, like for example Arg62 of the beta-wing (Fig. 3B), thereby impairing its function as a repressor of stress response genes. Identification of the CtsR/McsB arginine phospho-switch (Fig. 3C) creates new perspectives to enhance our understanding of prokaryotic and eukaryotic transcriptional regulation.