Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

Abstract

Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.AAA+ proteases | allosteric coupling | pathogen drug target

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

“…3B). These data support the conclusion that the stimulation of ATPase activity reflects a specific interaction and further suggest that only one heptameric ring in ClpP1P2 interacts with ClpC1 or ClpX, as was shown by Leodolter et al (36) and suggested by the binding of ADEP to only the ClpP2 ring in the ClpP1P2 structure reported by Schmitz et al (25). The addition of protein substrates for ClpX (GFP-SsrA) or ClpC1 (FITC-casein) to assays with ClpP1P2 Peptidase activity was measured with Ac-PKM-amc as described under "Experimental Procedures" but with varying concentrations of the activator Bz-Nva-Ile.…”

“…The two molecules differ primarily in the N-terminal region, where residues 15-32 of ClpP2 form a very stable ␤-hairpin that extends away from the body of the compact tetradecamer. As observed in a recent crystal structure of ClpP1P2 (25), the extended conformation is stabilized by interaction with the loop extensions from another ClpP2 molecule related by crystallographic symmetry. The first seven residues of ClpP1 are not visible and presumably form a flexible coil.…”

“…Similar effects were obtained with Bz-Nva-Ile (data not shown). In contrast, Z-Ile-Leu (Z-IL), the activator used by Schmitz et al (25) in their crystallographic and kinetic studies, produced weaker activation than both Bz-LL and Z-LL (Fig. 1).…”

“…However, the addition of dipeptides at concentrations in excess of those needed for activation led to competitive inhibition of peptidase activity, which suggested that activators might bind at the catalytic sites in addition to possible allosteric sites in ClpP. Binding of activators to the active site was confirmed recently by x-ray crystallography (25); however, a mechanism that could reconcile the binding of activators to the active sites with their ability to enhance peptidase activity remained to be defined. Previously, binding of acyldepsipeptide antibiotics (ADEPs) were shown to stabilize assembly of homomeric ClpP tetradecamers (26).…”

“…Also unlike those dipeptides, Bz-LL showed maximal activation at the high concentrations used for crystallization. While this work was in progress Schmitz et al (25) reported the structure of Mtb ClpP1P2 with the less potent peptide activator, benzyloxycarbonyl-Ile-Leu (Z-IL), bound at active sites. In addition, that structure included ADEP molecules bound to the docking site on ClpP used by the cognate ATPases, which complicated interpretation of the allosteric effects of the dipeptide activator alone.…”