Fate of pup inside the Mycobacterium proteasome studied by in-cell NMR

• Published in: PloS one Vol. 8; no. 9; p. e74576
• Main Authors: Maldonado, Andres Y, Burz, David S, Reverdatto, Sergey, Shekhtman, Alexander
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.09.2013; Public Library of Science (PLoS)
• Subjects: Adenosine triphosphatase; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biodegradation; Chemistry; Degradation; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression Regulation, Bacterial; Immune system; Microbial drug resistance; Models, Molecular; Molecular Sequence Data; Mycobacterium; Mycobacterium smegmatis; Mycobacterium smegmatis - genetics; Mycobacterium smegmatis - metabolism; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular - methods; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Proteasomes; Protein Binding; Protein Interaction Domains and Motifs; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Precursors - chemistry; Protein Precursors - genetics; Protein Precursors - metabolism; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Studies; Substrates; Tuberculosis; Ubiquitin; Ubiquitins - chemistry; Ubiquitins - genetics; Ubiquitins - metabolism; Virulence; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0074576

The Mycobacterium tuberculosis proteasome is required for maximum virulence and to resist killing by the host immune system. The prokaryotic ubiquitin-like protein, Pup-GGE, targets proteins for proteasome-mediated degradation. We demonstrate that Pup-GGQ, a precursor of Pup-GGE, is not a substrate for proteasomal degradation. Using STINT-NMR, an in-cell NMR technique, we studied the interactions between Pup-GGQ, mycobacterial proteasomal ATPase, Mpa, and Mtb proteasome core particle (CP) inside a living cell at amino acid residue resolution. We showed that under in-cell conditions, in the absence of the proteasome CP, Pup-GGQ interacts with Mpa only weakly, primarily through its C-terminal region. When Mpa and non-stoichiometric amounts of proteasome CP are present, both the N-terminal and C-terminal regions of Pup-GGQ bind strongly to Mpa. This suggests a mechanism by which transient binding of Mpa to the proteasome CP controls the fate of Pup.