ATP-dependent proteases of bacteria: recognition logic and operating principles

Abstract

ATP-powered AAA+ proteases degrade specific proteins in intracellular environments occupied by thousands of different proteins. These proteases operate as powerful molecular machines that unfold stable native proteins before degradation. Understanding how these enzymes choose the "right" protein substrates at the "right" time is key to understanding their biological function. Recently, proteomic approaches have identified numerous substrates for some bacterial enzymes and the sequence motifs responsible for recognition. Advances have also been made in elucidating the mechanism and impact of adaptor proteins in regulating substrate choice. Finally, recent biochemical dissection of the ATPase cycle and its coupling to protein unfolding has revealed fundamental operating principles of this important, ubiquitous family of molecular machines.

Figure 1

Substrate recognition, unfolding and degradation by an AAA+ protease. (a) The protease consists of a hexameric AAA+ unfoldase (blue) and a compartmental peptidase (yellow). The initial recognition step shown here is mediated by residues in the central pore of the AAA+ ring, which bind to a peptide tag sequence attached to an otherwise native protein substrate. Repetitive conformational changes in the AAA+ ring, driven by ATP hydrolysis, translocate the tag and attached protein through the pore. This vectorial movement unfolds the protein and transports the denatured polypeptide into the degradation chamber of the peptidase. Active sites in the peptidase chamber cleave the unfolded substrate into short peptides, which are subsequently released. (b) Binding of protein substrates to AAA+ proteases can be assisted by adaptor proteins. In this example, the ternary recognition complex is stabilized by interactions between the adaptor (black and purple) and the substrate, between a flexible tail of the adaptor and the AAA+ ring of the protease, and between the substrate tag and the pore of the AAA+ ring. The ternary complex is more stable than the binary complex shown in (a), enabling more efficient degradation at low substrate concentrations. Some adaptor proteins are also degraded, whereas others are resistant to proteolysis and can therefore participate in many rounds of substrate delivery.

Figure 2

Mechanisms of regulated substrate recognition. (a). When degradation tags (red) are part of the folded structure of a protein or complex, they can be inaccessible to AAA+ proteases. Such ‘hidden’ tags can be revealed by internal cleavage (left), unfolding (center), or complex dissociation (right). In these cases, protein turnover can be regulated by the reactions that lead to exposure of the degradation tag. (b) Macromolecular assembly can be required for efficient substrate recognition and degradation, when monomeric recognition signals are weak. Left, trimerization of a protein places a degradation tag (red) and two tethering peptides (cyan) in the proper geometry to interact simultaneously with the pore and tethering sites (light blue rectangles) of an AAA+ protease. Multivalent binding of this type can be orders of magnitude stronger than any of the individual binding reactions. Right, heteromeric assembly of an adaptor containing a tethering signal and a substrate containing a degradation tag is required for efficient substrate recognition.

Figure 3

The cost of degradation. For denatured substrates and native substrates that are easily unfolded, the cost of degradation is determined mainly by the cost of translocation. For some substrates of ClpXP, this cost is ∼1 ATP molecule for each residue degraded [37]. Thus, ∼100 ATP molecules would be hydrolyzed during degradation of a 100-residue protein. For native substrates that are difficult to denature, the cost of degradation can be much higher. When the native structure of the substrate is sufficiently stable to resist mechanical unfolding by the protease, the substrate is usually released [40]. In these instances, many cycles of binding, attempted unfolding and release can occur before enzymatic denaturation is successful, and the energetic cost of unfolding alone can exceed the hydrolysis of 500 molecules of ATP [37].