DegP 24-mers are spacious chaperones

A few weeks back I mentioned a paper in PNAS on allosteric regulation of DegP protease function by its PDZ domains. This week in Nature, the same group (same first author, even) provides some intriguing new insights into the workings of this combined chaperone and protease. Using different arrangements of a base trimer structure, DegP assembles into multimers of 6, 12, and 24 protein units. Krojer et al. determine the structures of the larger complexes using cryo-electron microscopy and X-ray crystallography, and how these structures might achieve the refolding and degradative functions of this HtrA protein family member.

As I mentioned last time, a single DegP protein consists of a protease domain and two PDZ domains, a domain I talk about a lot. This makes for a decently-sized protein, but in fact DegP is rarely encountered in vivo as a monomer. It is known to form trimers and hexamers, and now dodecamers and whatever fancy Latin or Greek word you would use for a 24-mer. In all of these cases what we're really dealing with are higher assemblies of trimers. For instance, the dodecamer is a tetramer of trimers. In addition to its ability to degrade proteins, DegP is known to have a chaperone function, and also to be able to shepherd OMPs (outer membrane proteins) through the periplasm of E. Coli.

In the case of the 24-mer, the DegP molecules assemble into a giant, hollow octahedral shape with an interior cavity 110 Å wide, which is larger than the cavity of the well-known chaperone GroEL. The PDZ domains mediate contact between adjacent trimers, and the protease domains form the "faces" of the octahedron. From the first figure in the paper it almost looks like you could cram 2 folded OMP proteins into the cavity formed by this structure. This oligomeric complex is so large it could conceivably span the whole periplasmic space between the inner and outer membranes of an E. Coli. Because the 24-mer has fairly large pores, it seems possible that it could form a tunnel that protects OMPs from aggregation and degradation as they cross the periplasm. In addition, positively-charged residues concentrated on the surfaces of the PDZ domains appear to give the multimer some affinity for membranes; these positive charges are concentrated around the edges of the pores.

To check this idea in vivo, Krojer et al. made a DegP-null strain of bacteria and measured the concentration and location of OMPs. They found that for several OMPs, deleting DegP did not change the concentration of the OMP in a whole cell lysate, but reduced levels of these proteins in the outer membrane. Further experiments indicated that DegP oligomers can protect OMPs from proteases. DegP itself can degrade unfolded OMPs, but stabilizes the folded proteins.

The researchers also managed to catch a glimpse of an OMP inside a DegP oligomer. In the case of the 24-mer this was difficult, probably because the sheer size of the enclosed space allows so many orientations of the OMP that getting a regular structure is impossible. However, they found that the structure of DegP dodecamers bound to OMP was fairly homogeneous, allowing an investigation by cryo-EM. You can see an image of the structure (shamelessly stolen from Figure 5) at left; the DegP molecules are in warm tones, and a molecule of OmpC is visible in blue. Again, the protease domains form the faces of this tetrahedral cage, while the PDZ1 domains (not PDZ2 in this case) mediate trimer-trimer contacts.

But what about DegP's protease function? Chromatography experiments (Figure 2) suggest that at room temperature, the presence of unfolded substrate molecules induces the formation of higher-order oligomers. These experiments cannot tell us whether these 12- and 24-mers have the same conformations as determined in the experiments above, but the fact that the PDZ1 domains mediate trimer-trimer contacts in both complexes suggests a possible mechanism for the allosteric activation noted previously. However, at higher temperatures where protease activity is markedly increased, the dominant species in solution was the trimer itself, even in the presence of substrates.

The authors propose that the hexameric form of DegP is a resting state, and that DegP₁₂ and DegP₂₄ form in response to specific stimuli. This model certainly fits the observations in solution, but it seems possible to me that the presence of membranes could induce formation of DegP₂₄. In vivo studies using fluorescence or TEM may be able to address what form actually predominates in the periplasm. Additionally, these results do not directly address the role of oligomerization in the switch between proteolytic and chaperone function. Particularly crucial in this regard is the question of whether the larger complexes that form during proteolysis are the same as those that form around OMPs. While it's reasonable to think that they are, the demonstrated structural versatility of DegP trimers suggests that these large assemblies may represent alternative conformations. Alternatively, it could be that features of the periplasm make DegP₂₄ and DegP₁₂ poor proteases in bacteria, and that the more rapidly diffusing naked trimer is the only efficient protease in vivo among DegP oligomers.

If, however, the chaperone and protease oligomers are structurally equivalent, a whole new class of questions opens up. Is formation of 12- and 24-mers sufficient to activate proteolysis, or is some additional step required? What protects folded OMPs from degradation by the protease subunits? These are challenging questions, but the new structures will be of great assistance in guiding the design of the genetic and biochemical experiments that answer them.

1. Krojer, T., Sawa, J., Schäfer, E., Saibil, H.R., Ehrmann, M., Clausen, T. (2008). Structural basis for the regulated protease and chaperone function of DegP. Nature, 453(7197), 885-890. DOI: 10.1038/nature07004