Abstract
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.
Highlights
Inside a cell, macromolecular complexes are assembled along specific pathways necessary to carry out biological functions in the presence of a crowded cytosol [1,2,3]
In STINT-NMR, protein over-expression is induced in labeling medium to produce a uniformly labeled [U-15N] target protein containing NMR-active nuclei; cells are transferred to non-labeling medium to induce overexpression of the interactor protein. 15N- or 13C- edited heteronuclear single quantum coherence (1H{15N}-HSQC or 1H{13C}-HSQC) NMR experiments are performed to monitor the chemical shifts of target backbone amide or side chain 13C-1H
To determine if intracellular Pup contains any regions of induced secondary structure due to macromolecular crowding, [U-15N] Pup-GGQ was over-expressed in E. coli and an in-cell 1H{15N}-HSQC NMR spectrum was collected (Figure 1A)
Summary
Macromolecular complexes are assembled along specific pathways necessary to carry out biological functions in the presence of a crowded cytosol [1,2,3]. Often, during assembly, effector molecules such as ligands or substrates are present. One method to study macromolecular complexes inside a cell that affords temporal control over assembly is STINT-NMR [5,6,7]. In STINT-NMR, protein over-expression is induced in labeling medium to produce a uniformly labeled [U-15N] target protein containing NMR-active nuclei; cells are transferred to non-labeling medium to induce overexpression of the interactor protein. The order of sequential over-expression of target and interactor proteins can be reversed, allowing temporal control over the assembly of the complex
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