AAA+ Machines of Protein Destruction in Mycobacteria

Abstract

The bacterial cytosol is a complex mixture of macromolecules (proteins, DNA, and RNA), which collectively are responsible for an enormous array of cellular tasks. Proteins are central to most, if not all, of these tasks and as such their maintenance (commonly referred to as protein homeostasis or proteostasis) is vital for cell survival during normal and stressful conditions. The two key aspects of protein homeostasis are, (i) the correct folding and assembly of proteins (coupled with their delivery to the correct cellular location) and (ii) the timely removal of unwanted or damaged proteins from the cell, which are performed by molecular chaperones and proteases, respectively. A major class of proteins that contribute to both of these tasks are the AAA+ (ATPases associated with a variety of cellular activities) protein superfamily. Although much is known about the structure of these machines and how they function in the model Gram-negative bacterium Escherichia coli, we are only just beginning to discover the molecular details of these machines and how they function in mycobacteria. Here we review the different AAA+ machines, that contribute to proteostasis in mycobacteria. Primarily we will focus on the recent advances in the structure and function of AAA+ proteases, the substrates they recognize and the cellular pathways they control. Finally, we will discuss the recent developments related to these machines as novel drug targets.

Keywords: AAA+ protease complexes; Mycobacterium; novel drug targets; proteasome; protein degradation.

Figure 1

Linear cartoon of the different AAA+ proteins in mycobacteria, illustrating the position of various domains and motifs. The AAA+ domains either belong to the classic (light blue) or HCLR (dark blue) clade. Each AAA+ domain contains a consensus sequence for ATP binding (GX4GKT/S, where X is any amino acid) and hydrolysis (hDD/E, where h is any hydrophobic amino acid) known as the Walker A (A), and Walker B (B) motifs, respectively. Most AAA+ proteins contain an unique accessory domain, such as the zinc-binding domain (ZBD, in pink) in ClpX, the Clp N-terminal domain (orange) in ClpC1 and ClpB, the Lon SB (substrate binding) domain (green) in Lon, the α-helical (yellow) and OB/ID (pink) domains in Mpa, the p97 N-terminal domain (black) in Msm0858 and the Tetratricopeptide (TPR)-like domain (gray) in VCP-1. ClpC1 and ClpB also contain a middle (M) domain (yellow) located between the first and second AAA+ domain. The membrane-bound AAA+ protein, FtsH contains two transmembrane domains (black bars) separated by an extracellular domain (ECD, in white) and a C-terminal metallopeptidase (M14 peptidase) domain (red) containing the consensus sequence (HEXGH). Lon contains an N-terminal substrate binding (Lon SB) domain a central AAA+ domain and a C-terminal serine (S16) peptidase domain (red) with the catalytic dyad (S, K). All cartoons are derived from the sequences for the following M. smegmatis proteins ClpX (A0R196), ClpC1 (A0R574), FtsH (A0R588), Lon (O31147), Mpa (A0QZ54), ClpB (A0QQF0), p97/Msm0858 (A0QQS4), VCP-1/Msm1854 (A0QTI2). Domains (and domain boundaries) were defined by InterPro (EMBL-EBI) as follows: AAA+ (IPR003593); C4-type Zinc finger (IPR010603); Clp N-terminal (IPR004176); UVR or M (IPR001943); Lon SB (substrate binding) (IPR003111); p97 N-terminal (IPR003338); p97 OB/ID (IPR032501); Tetratricopeptide (TPR)-like (IPR011990); S16 protease (IPR008269), M41 protease (IPR000642).

Figure 2

In the first step, the substrate (green) engages with the AAA+ unfoldase (blue) via the degradation tag (commonly referred to as a degron). The degron (purple) is generally located at the N- or C-terminal end of the substrate, although in some case it may be internal (and exposed following unfolding or dissociation of the protein from a complex). For direct recognition by the AAA+ unfoldase (blue), the degron is engaged either by a specialized accessory domain or by specific loops, located at the distal end of the machine. Following recognition of the degron, the substrate protein is unfolded by the ATP-dependent movement of axial pore loops. The unfolded substrate is then translocated into the associated peptidase (red), where the peptide bonds are hydrolyzed by the catalytic residues (black packman) into short peptides. The peptides are released, either through the axial pore or holes in the side walls that are created during the cycle of peptide hydrolysis.

Figure 3

In the presence of the dipeptide activator (z-LL), ClpP1 (orange), and ClpP2 (red) form either homo- (left) or hetero-oligomeric complexes (middle). Activator binding is essential for propeptide processing of both ClpP proteins in Mtb (while only ClpP1 is processed in Msm). Hetero-oligomeric complexes are activated (black packman) through the complementary docking of Phe147 (F) of ClpP1, into a pocket on the handle of ClpP2. In contrast, homo-oligomeric complexes lack this complementary docking and are not active. The unfoldase (blue) docks only to a single face of the active peptidase (i.e., ClpP2) to generate an asymmetric machine. ADEP docks only to the hydrophobic pockets of ClpP2 and as such prevents docking of the unfoldase component.

Figure 4

Seven α-subunits (purple) first assemble into a heptameric ring (α-ring), which is used as a template to form a half-proteasome, by assembly of the β-subunits into a heptameric ring (on the α-ring template). Next, two half-proteasomes assemble, triggering removal of the N-terminal propeptide of the β-subunits and activation of the 20S CP. Finally, the C-terminal QYL motif of an activator (blue) such as Mpa or PafE/Bpa docks into a hydrophobic pocket on the α-ring of the proteasome, which triggers “gate-opening” of the N-terminal peptides thereby allowing access of substrates into the catalytic chamber of the protease.

Figure 5

The 20S CP interacts with two different activators, both of which contain a QYL motif at the C-terminus to trigger “gate-opening” of the α-ring of the proteasome. Mpa (dark blue) is an ATP-dependent activator of the 20S CP (top panel). The ring-shaped hexamer is composed of three domains, a coiled-coil (CC) domain for interaction with pupylated substrates, an oligosaccharide/oligonucleotide-binding (OB) domain which stabilizes the hexamer and an AAA+ domain which uses the hydrolysis of ATP to drive unfolding of the pupylated substrate. The second activator (Bpa/PafE) is an ATP-independent dodecamer (light blue), which triggers “gate-opening” of the α-ring pore, by docking into the hydrophobic pockets on the surface of the α-ring. The ring-shaped dodecamer contains a wide (~40 Å) hydrophobic channel, which is proposed to interact with hydrophobic (Hy) residues that are exposed in proteins such as HspR (heat-shock protein R) and model unfolded proteins.

Figure 6

Mechanism of action of different Clp protease inhibitors and activators. (A) ClpP dysregulators such as ADEP (green circle) dock into the hydrophobic pocket of ClpP2, where they (1) activate the protease to trigger uncontrolled degradation of cellular proteins and (2) inhibit ATPase docking thereby preventing the regulated turnover of specific substrates that are delivered to the protease by the ATPase. (B) β-lactones (blue triangle) inhibit ClpP by inactivating the catalytic Ser (black packman) residue of the protease. (C) ClpC1 dyregulators such as CymA (pink circle), ecumicin (orange hexagon), or lassomycin (orange hexagon) bind to the N-terminal domain of ClpC1, accelerating its ATPase activity. In the case of CymA, docking to the N-terminal domain prevents movement of the domain, which triggers the accelerated turnover of proteins. In contrast, ecumicin and lassomycin uncouple ClpC1 from the peptidase, thereby preventing the regulated turnover of specific proteins.