AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control

Abstract

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA þ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97

“…Conversely, the specific inhibition of the ATPase activity of p97 by the N terminus of UBXD1 supports the notion that the intrinsic features of UBXD1-N also apply in the context of the full-length protein. Although the substoichiometric inhibition of p97 by UBXD1-N is quite unusual, such phenomena have already been described for oligomerizing protomers (53)(54)(55)(56)(57). Therefore, we hypothesize a kind of sustainability of the UBXD1-N interaction with the p97 hexamer.…”

Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

“…[36][37][38] Beta-lactones, the only specific ClpP inhibitors reported to date, have been applied as tools to probe the binding pocket and mechanism of inhibition, as well as the transient stability of the ClpP tetradecamer and its conformational switching. 23,25,39 One additional consequence of beta-lactone-based ClpP inhibition is the reduction of virulence in pathogenic bacteria such as MRSA and the eradication of Mycobacteria that express two essential ClpP isoforms. 19,20,40 Here, we expand the set of ClpP tools through a novel class of phenyl esters that surpass beta-lactones in terms of potency in peptidase and protease inhibition, reaction kinetics, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 17 target selectivity in the soluble fraction of the proteome, plasma half life, acyl-enzyme stability and de-oligomerization as the preferred mechanism of inhibition.…”

PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

“…Finally, the roles that bacterial Clp proteins play in cell division, stress response and pathogenicity (Frees et al, 2014), have placed them at the center of several drug discovery programs (Böttcher and Sieber, 2008; Brötz-Oesterhelt and Sass, 2014; Conlon et al, 2013; Gersch et al, 2015; Hackl et al, 2015). Our data demonstrate that targeting the P. falciparum plastid-localized Clp proteins is a viable strategy for antimalarial drug development and future work will allow us to repurpose highly active antibacterial compounds as effective anti-malarial agents.…”