PDZ domains allosterically regulate the bacterial envelope protease DegP

Anyone who reads this blog regularly knows that I have a great deal of interest in the allosteric potential of the PDZ domain, a small protein binding domain that can be found in every branch of the tree of life. In previous posts I've discussed the evidence for allosteric communication within the PDZ domain, as well as evidence for long-range energetic interactions. An upcoming paper emerging from a collaboration of several European groups provides yet another example of the PDZ domain's allosteric potential bearing fruit, in this case in the regulation of a protease. The article is open-access, so go ahead and open it up in another window to follow along.

Krojer et al. are investigating the heat response in bacteria. Just as your body responds to ambient warmth (by sweating, etc.), bacteria have several systems to help them cope with high temperatures. These include systems for folding proteins that have lost their shape due to the heat, and also systems that degrade proteins when the refolding system can't keep up. A protein that breaks down other proteins is called a protease, and enzymes of this type are widely used in nature (blood clotting, viral maturation, digestion, etc.). Because of their destructive potential proteases are often tightly regulated, as is the case with the protease in this article, DegP. In fact, DegP can also serve as a refolding protein (or chaperone)—some regulatory mechanism causes a switch between these functions. DegP is an E. coli enzyme, but has a similar architecture to some human enzymes linked to diseases that involve protein misfolding and aggregation.

Krojer et al. performed experiments to characterize the proteolytic activity of DegP, mostly summarized in Figure 1. These results indicate that DegP is processive, i.e. that it makes many cuts on a target rather than just one. Using mass spectrometry the researchers determined that the targets got cut up into chunks 8-22 residues long, with the most common lengths being 12 and 17 residues. The protease preferred to cut proteins after a valine, alanine, isoleucine, or threonine: these are very common residues in proteins and interestingly most are β-branched. However, when they made short peptides that matched the apparent cleavage pattern, they did not observe any proteolysis.

From this the authors concluded that binding to the PDZ1 domain of DegP was necessary for the proteins to get cut. They performed a series of experiments with longer peptides that showed that a proper binding site 13-17 residues away from the cleavage site was necessary to get proteolysis. Also, the C-terminal residues preferred by PDZ1 are the same as the ones where the protease cleaves. Modeling an unstructured peptide substrate into the known structure of the DegP complex (Figure 4) indicates that a peptide chain ~16 residues long is needed to reach from the PDZ1 domain of one DegP to its own protease domain, and a chain ~12 residues long is needed to stretch to the protease site of an adjacent DegP molecule.

Well, this suggests a tidy little model. The C-terminus of an unfolded protein binds at the PDZ domain and is cut by the protease about 12 or 16 residues down the line. The cut produces a new C-terminus, which binds at the PDZ domain, and the process repeats. In this way the DegP complex processively degrades unfolded proteins and the bacteria are saved from toxic aggregates. But this is not the whole story. You see, it turns out that DegP is pretty efficient at cutting peptides even if the PDZ substrate and the cleavage site are not attached to each other.

The above model suggests two predictions. First, a peptide that binds to the PDZ1 domain (ALE peptide) but cannot be cleaved by the protease should inhibit the proteolysis of an unfolded protein. Second, the activity of DegP towards a substrate that is too short should not be affected by adding an ALE peptide. However, when the researchers in this case performed these experiments the results were quite different than expected. The ALE peptide slightly activated the degradation of an unfolded protein, and enhanced the cleavage of the short peptide 50-fold. Further experiments indicated that the binding of a peptide to the PDZ1 domain of one DegP molecule activated the protease of that molecule and one neighboring molecule of the complex.

So now the model is more complex, but also much more interesting. Binding of an unfolded protein's C-terminus to the PDZ1 domain does help generate the processivity and molecular ruler effects mentioned previously. But this binding also allosterically increases the intrinsic rate of proteolysis in nearby protease subunits. The effect is a sort of positive feedback mechanism that keeps the protease running until the whole target protein is degraded. This adaptation means that the protease can activate rapidly when there are a lot of free termini floating around, but won't go chopping up every flexible loop it comes across.

The precise mechanism by which binding at the PDZ site activates the protease is beyond the scope of the present study. It may be that the networks suggested by previous research do not come into play in this instance. Looking at the biological complexes predicted from the crystal structure (explore it at the PDB) I would guess that binding-dependent remodeling of the strand linking the protease domain to PDZ1 may be responsible, rather than the dynamic networks. Future experiments will probably address this allosteric mechanism.

1. Krojer, T., Pangerl, K., Kurt, J., Sawa, J., Stingl, C., Mechtler, K., Huber, R., Ehrmann, M., Clausen, T. (2008). Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins. Proceedings of the National Academy of Sciences 105(22), 7702-7707 DOI: 10.1073/pnas.0803392105 OPEN ACCESS