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

Abstract

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP 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 of Mtb ClpC1 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 ␤-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.

Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis

“…Indeed, despite the low substrate affinity compared to ClpX [21], ClpC1 in association with ClpP1P2 in the presence of 2.5 mM of the BzLL activator could catalyze the degradation of GFPssra with an apparent K M of 11.3 ± 1.5 μM GFPssra (Fig1a). The ATP dependence for ClpC1P1P2 GFPssa degradation displayed evidence of cooperativity with half maximal activity at 1.4 ± 0.1 mM (Fig1b, 1c), a value significantly lower than the one previously described for ClpC1 ATPase activity [5,21]. …”

“…Previous reports have shown that ClpC1 is able to recognize intrinsically unfolded proteins with exposed hydrophobic patches [5]. As described for other members of the AAA+ family [27], the recognition of these proteins by ClpC1 is likely different from the recognition and degradation of tightly folded proteins, such as GFPssra [27].…”

“…The standard protocol for 384 plates (Corning 3184) consisted of 2 steps (unless otherwise notices all concentrations refer to the final volume of 15 μl). A first step where ClpC1 monomer (2.5 μM), ClpP1P2 monomer (10 μM), the activator Bz-LL (2.5 mM) [5], FITC-casein (5.5 μM) in HEPES pH 7.6 (50 mM, Sigma), KCl (100 mM, Sigma), Triton X-100 ( 0.22 mM, Sigma) and MgCl 2 (40 mM, Sigma) in total volume of 10 μl were incubated for at least 15 minutes with the test compound (max DMSO concentration of 4%). In a second step ATP (10 mM, buffered to pH 7.6) or water (column 18) were added.…”

“…While neither ClpP1 nor ClpP2 by itself has proteolytic activity, when mixed together in the presence of a dipeptide activator, they form the active complex containing one ClpP1 and one ClpP2 ring. Whereas the mechanism of this activation is still unclear, Xray structures of the complex in the presence of the activator Benzoyl-Leu-Leu (Bz-LL), an N-terminally blocked dipeptide, show that it binds in opposite orientations in ClpP1 and ClpP2 [5]. While in ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket.…”

“…While 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 strands [5]. …”