title: 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, 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.
authors: From the Macromolecular Crystallography Laboratory, NCI, National Institutes of Health and Basic Research Program, Leidos Biomedical Research, Frederick National Laboratory, Frederick, Maryland 21702.
authors: Mi
authors: M
authors: Li
authors: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and.
authors: Olga
authors: O
authors: Kandror
authors: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and.
authors: Tatos
authors: T
authors: Akopian
authors: the Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors: Poorva
authors: P
authors: Dharkar
authors: From the Macromolecular Crystallography Laboratory, NCI, National Institutes of Health and.
authors: Alexander
authors: A
authors: Wlodawer
authors: the Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 mmaurizi@helix.nih.gov.
authors: Michael R
authors: MR
authors: Maurizi
authors: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and Alfred_Goldberg@hms.harvard.edu.
authors: Alfred L
authors: AL
authors: Goldberg
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D009097:Multienzyme Complexes; C119658:ClpP2 protein, bacteria; D012697:Serine Endopeptidases
keywords: enzyme mechanism; protease; protein complex; protein degradation; x-ray crystallography