Abstract
The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, inMycobacterium tuberculosis(Mtb) are essential and, therefore, promising drug targets. TheMtbClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity ofMtbClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.
Highlights
The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, promising drug targets
Bz-Leu-Leu and Bz-Nva-Ile Are Potent Activators of Mtb ClpP1P2—In our earlier studies [11] we found that Z-LL stimulated formation of the active the Mtb ClpP1P2 complex but that maximal activation required 5 mM activator and no activity was evident below 2 mM (Fig. 1)
When peptidase activity was measured with the preferred fluorogenic substrate, AcPKM-amc [11, 13], a half-maximal rate of hydrolysis was obtained at ϳ1.5 mM Bz-LL, and maximum stimulation occurred at 2.5 mM (Fig. 1)
Summary
M. tuberculosis; eClpA, E. coli ClpA; Z-LL, benzyloxycarbonyl-Leu-Leu; Bz-LL, benzoyl-Leu-Leu; ADEP, acyldepsipeptide; Bz-Nva-Ile, benzoyl-norvaline-isoleucine; Z-IL, benzyloxycarbonyl-IleLeu; Ac-PKM-amc, acetyl-PKM-7-amido-4-methylcoumarin; Bis-Tris, 2[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ATP␥S, adenosine 5Ј-O-(thiotriphosphate). Benzyloxycarbonyl-Leu-Leu (Z-LL) was the most effective activator from among a group of related blocked dipeptides and dipeptide derivatives We showed that these molecules activated formation of ClpP1P2 complexes by promoting the dissociation of the inactive homotetradecameric ClpP1 and ClpP2 complexes into heptameric rings and that association into the heterotetradecameric ClpP1P2 complex increased peptidase activity Ͼ1000-fold [11]. Studies to date with Mtb ClpP1P2 have involved recombinant proteins expressed in heterologous systems, and it is unclear whether dipeptide or analogous activator mechanisms are required for active enzyme formation in vivo. ClpP1P2 with Bz-LL bound enhanced ATP hydrolysis by Mtb ClpX and ClpC1 and ATP-dependent degradation of model protein substrates These findings make it very likely that the structure described here is the physiologically active form of the ClpP1P2 complex
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