Abstract
Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.
Highlights
Mycobacterium tuberculosis (Mtb) is a gram-positive bacterium of the phylum Actinobacteria and the causative agent of tuberculosis
While first theories proposed that each of the two chaperones, ClpX and ClpC1, binds to either ClpP1 or ClpP2, our results clearly show that both chaperones only use the ring surface of ClpP2 to build the protein degradation-competent complexes
There are two motifs on the ClpP protease core involved in chaperone interaction, the Nloop and a hydrophobic surface patch [14, 17,18,19,20,21]
Summary
Mycobacterium tuberculosis (Mtb) is a gram-positive bacterium of the phylum Actinobacteria and the causative agent of tuberculosis. The Clp chaperoneprotease is a bacterial multi-subunit protein complex involved in intracellular protein degradation. It is active in general protein quality control as well as specific degradation of proteins. The Clp chaperone-proteases form cylindrical complexes built from rings of protease and chaperone subunits stacked on top of one another. The other interaction feature is a hydrophobic patch located on the face of the protease ring to which binds a loop of the chaperone containing a conserved LGF-motif in case of the Mtb chaperones ClpX and ClpC1 (LGF-loop) [14, 20, 21]. Antibiotics of the acyldepsipeptide (ADEP) class bind to this patch in place of the LGF-loop and deregulate the Clp protease by mimicking chaperone binding [22,23,24]
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