Fig 1.
Binding of wild-type Mpa to open-gate proteasome.
Open-gate Mtbproteasome (oCP) at concentrations from 16 μM to 4 nM was titrated against 5 nM fluorescein-labelled MtbMpa and their association probed with Microscale Thermophoresis (MST) in three replicates. The dependence of the bound fraction of Mpa (from normalized fluorescence signal change) on the proteasome concentration was fitted with the company-provided analysis software, yielding a dissociation constant of 0.52 ± 0.05 μM.
Fig 2.
Association of four different pupylated substrates or free Pup to Mpa probed by microscale thermophoresis.
(A) Binding of Pup-substrate to wild type Mpa in the absence of nucleotide. Data was acquired with MST in 4–8 replicates, by titrating proteins from 7 μM to 3 nM against their labeled binding partner. For pupylated PanB, PckA and Icl1, fixed concentrations of Pup-substrate-Fl were used (70, 40 and 150 nM protomer) and Mpa was titrated to the reaction, while for Pup-FabD3KR, the substrate was titrated against 5 nM of labeled Mpa hexamer for a better signal to noise ratio. The dependence of the bound fraction of fluorescein-labeled molecule (from normalized fluorescence signal change) on the concentrations of binding partner was fitted with the company-provided analysis software, yielding dissociation constants of 0.24 ± 0.06 μM for Pup-FabD3KR, 0.17 ± 0.03 μM for Pup-PanB, 0.21 ± 0.10 μM for Pup-PckA and 0.16 ± 0.03 μM for Pup-Icl1. (B) Binding of PupE to wild type Mpa in the absence of nucleotide. Data was acquired with MST in 5 replicates, by titrating Pup from 60 μM to 40 nM against 5 nM fluorescently labeled Mpa hexamer. Fitting of the thermophoresis binding curve with the company-provided analysis software yielded a dissociation constant of 3.31 ± 0.65 μM. (C) Purified pupylated substrates analyzed on Coomassie stained SDS-PA gel. The target proteins used in this study were heterologously expressed in E. coli, purified and subsequently pupylated in vitro at 3 μM concentration with 5 molar equivalents His10-3C-MtbPupE and 1/3 molar equivalents MtbPafA, before immobilized metal affinity chromatography (IMAC) to ensure clean and homogeneous sample. While FabD3KR, PanB and PckA could be pupylated at every protomer, the homotetrameric Icl1 had only two out of four subunits pupylated.
Fig 3.
Michalis-Menten analysis for the degradation of Pup-substrate by the Mpa-oCP complex.
Turnover numbers (min-1 per Mpa-oCP complex) were calculated from the initial phase (within ≤ 10% signal change) of fluorescein-labeled Pup-substrate degradation at excess substrate concentrations. With the exception of Pup-PanB-Fl, pupylated substrates were used in a ratio of pupylated substrate protomer to Mpa of 1:10. Pup-PanB-Fl was used in a ratio of substrate protomer to Mpa of 1:100. The monomeric FabD3KR and PckA and the decameric PanB were thus in 10-fold excess over Mpa hexamer, the tetrameric Icl1 in 5-fold excess. Degradation curves were measured exciting at λex = 485 nm and recording the emission at λem = 525 nm in a plate reader. TONs were plotted with a confidence interval of 2x standard deviation against the corresponding concentrations of Pup-substrate-Fl with subsequent fitting to the Michaelis-Menten equation: TON = (kcat * [S]) / (KM + [S]). The kcat values for the reactions range between 0.14 and 0.31 min-1, while the KM values fall into a narrow, low nanomolar range (4–10 nM) (Table 1).
Table 1.
Kinetic parameters for degradation of four different Pup-substrates by the Mpa-oCP complex.
Fig 4.
Pupylome production and degradation.
(A) Mtb and Msm Pup share high sequence identity. While a few residues differ between MtbPup used for all experiments in this study and MsmPup, both the ATPase and ligase interaction segments, as well as the N-terminal sequence serving as threading element for engaging the Mpa pore are identical. (B) Flow-scheme for pupylome production and degradation. Endogenous PafA in M. smegmatis Δdop (MsmΔdop) cells was used to modify substrates in vivo with Strep-TEV-His6-MtbPupE, overexpressed from a plasmid. Pupylated substrates were purified via the Strep-tag on Pup. After proteolytic cleavage of the purification tag, purified wild-type Mpa and oCP were added to the reaction and samples were taken at the indicated time-points. Pupylated proteins remaining in the mixture were separated from secondary binders and the Mpa-oCP degradation complex via the His6-tag on Pup under denaturing conditions in 6 M guanidinium chloride. After protein precipitation with TCA/NaDoC, the samples were dissolved in SDS loading buffer and analyzed with SDS-PAGE and Western-blotting (Fig 5).
Fig 5.
Degradation time-course of purified pupylome by the Mpa-oCP complex.
Affinity-purified pupylome from Msm was subjected to degradation by the MtbMpa-oCP complex along a 24 hour time course. Aliquots drawn along the time-course are visualized by Coomassie-stained SDS-PA gels (PAGE) and anti-Pup Western-blotting (WB). During the sampling time-course, almost all of the pupylated substrates from the mixture are degraded in an evenly distributed fashion (two left panels). Only three major bands stay visibly behind on the Coomassie-stained SDS-PA gel at roughly 150, 120 and below 70 kDa (indicated with a star), on Western blot three equivalent bands can be detected, although the two at 150 and 120 kDa are faint. The control without added MtbMpa-oCP shows no loss of signal over the time of sampling.
Fig 6.
Michalis-Menten analysis of the Mpa-oCP dependent degradation of the three Pup-substrates identified in the pupylome degradation sample.
(A) Purified, in vitro pupylated Msm substrates analyzed on Coomassie stained SDS-PA gel. The heterologously expressed target proteins were purified and subsequently pupylated in vitro at 3 μM concentration with 5 molar equivalents His10-3C-MtbPupE and 1/3 molar equivalents MtbPafA followed by IMAC purification. The putative homodimers MSMEG_2412 and MSMEG_5049 are pupylated to 50%, likely reflecting pupylation of one protomer in each dimer. The dimeric MSMEG_1807 also exhibits pupylation of one protomer, but shows a more complex band pattern, since about half of the protomers of the unpupylated starting material were truncated. The full-length, unpupylated monomer signal is marked with an asterisk. (B) Fluorescence-based, real-time degradation assay of the three Pup-substrates identified in the pupylome degradation sample. Turnover numbers (min-1 per Mpa-CP) with a confidence interval of 2x standard deviation were plotted against the corresponding concentration of Pup-substrate-Fl and the curves were fitted to the Michaelis-Menten equation: TON = (kcat * [S]) / (KM + [S]).
Table 2.
Kinetic parameters for Mpa-oCP dependent degradation of the three Pup-substrates identified in the pupylome degradation sample.
Fig 7.
Depupylation time courses of Pup-substrate conjugates.
Pupylated substrate (2 μM pupylated protomer) is completely turned over by 0.2 μM Dop in a time frame ranging from one hour to more than eight hours. Pup-FabD3KR takes little above 1 hour, while pupylated PanB, MSMEG_1807 and MSMEG_5049 are depupylated within 2 hours. MSMEG_2412 takes about 5 hours for the same amount of pupylated protomers, and PckA as well as Icl1 are not fully depupylated even within eight hours.
Fig 8.
Binding of Pup-substrate conjugates to Dop in absence of nucleotide.
Data was acquired with MST in a minimum of three replicates, by titrating pupylated substrate from 10 μM to 4 nM against fluorescently labeled Dop. The dependence of the bound fraction of fluorescein-labeled molecule (from normalized fluorescence signal change) on the concentrations of binding partner was fitted with the company-provided analysis software, yielding dissociation constants of 17 ± 3 nM for Pup-PanB, 129 ± 41 nM for Pup-Icl1, 339 ± 55 nM for Pup-MSMEG_1807, 114 ± 25 nM for Pup-MSMEG_2412 and 121 ± 28 nM for Pup-MSMEG_5049.