Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

Abstract

Background: ClpP1P2 is a novel protease complex essential for viability of Mycobacterium tuberculosis. Results: Cleavage preferences of ClpP1P2 were defined, which allowed us to design potent substrate-based boronate inhibitors showing anti-mycobacterial activity. Conclusion: Excellent new fluorogenic peptide substrates of ClpP1P2 were obtained, and novel enzyme properties were identified. Significance: Selective inhibition of ClpP1P2 activity is a promising approach for drug development.

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

“…The activator was bound with the C terminus directed toward the catalytic residues in ClpP1. This orientation is similar to that expected for a degradation product immediately after cleavage of the acyl enzyme intermediate, which would allow the activator to be easily displaced by incoming tripeptide substrates in ClpP1, which mutagenesis has shown has almost all of the peptidase activity (13). If the Bz-LL were not easily displaced by incoming tripeptides in ClpP2, which lacks peptidase activity, the ClpP1P2 complex would persist and remain in the active conformation.…”

“…Bz-LL binding to ClpP2 does not mimic substrate binding, but it induces a change in the access channel to enhance peptide entry. This unexpected orientation of the activator in ClpP2 seems to explain the finding that ClpP1 is responsible for the peptidase activity, whereas ClpP2 exhibits little or none (13,25). We propose that Bz-LL binding to ClpP2 opens the axial channel of the complex so that substrates can be cleaved at the active site in ClpP1.…”

“…As shown here, the nature of the activating peptide and its concentration are critical for these interactions and the amount of activity. Both ClpP1 and ClpP2 have the conserved catalytic triad found in other ClpPs, and both proteins are essential for ATP-dependent proteolysis with ClpC1 or ClpX (11,13).…”

“…Furthermore, the activity and integrity of the complex are lost very rapidly upon removal of the activator by dilution. The presence of an activator is also needed to assemble active complexes of ClpP1P2 with ClpC1 or ClpX (11,13), and the enhanced ATP hydrolysis and ATP-dependent protein degradation were also lost immediately upon dilution into the buffer lacking the activator. This unusual requirement for activators appears to be unique to ClpPs from Actinobacteria, because in other species that contain two ClpP subunits, such as Listeria monocytogenes, formation of the active enzyme does not show a similar requirement (51).…”

“…ClpP1 and ClpP2 are essential for viability of M. tuberculosis, as are the regulatory ATPases, ClpX and ClpC1 (21), although the proteins that are targeted by the Clp protease complexes have not been identified (22). Novel antibiotics that prevent ATP-dependent proteolysis by ClpC1P1P2 (23,24) and compounds that block ClpP1P2 (13) have been shown to selectively kill M. tuberculosis. Consequently, the components of the Clp protease complexes are of special interest as potential targets for new classes of antimicrobial compounds.…”