McsB forms a gated kinase chamber to mark aberrant bacterial proteins for degradation

Abstract

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labeling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labeling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, unlike these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.

Keywords: B. subtilis; biochemistry; chemical biology; mass photometry; molecular biophysics; protein phosphorylation; protein quality control; self-compartmentalization; structural biology; targeted protein degradation.

Figure 1.. McsB_BS forms higher order oligomers in vitro and in vivo.

(a) Schematic picture of the McsB dimer, emphasizing its domain architecture and the location of the catalytic and allosteric sites (PD, phosphotransferase domain; DD, dimerization domain). (b) Size exclusion chromatography (SEC) of recombinant McsB_BS and McsB_GS. Triangular markers indicate the protein size at the peaks. (c) SEC analysis of lysates of heat-shocked and non-heat shocked B. subtilis cell cultures. Comparing the western blots to SDS-PAGE gels of isolated McsB dimer and HOO reveals the different size distributions of McsB under the applied conditions.

Figure 1—figure supplement 1.. Degradation pathways in comparison.

Comparison of the ubiquitin-proteasome and pArg-ClpCP degradation pathways that are similarly organized but use distinct degradation signals (orange).

Figure 1—figure supplement 2.. Higher order oligomers of McsB_GS.

Size exclusion chromatography of recombinant McsB_GS showing two dominant peaks (left panel). Secondary SEC runs of the two major peaks (right panel).

Figure 2.. Crystal structure of the McsB_BS octamer.

(a) Ribbon plot of the McsB_BS octamer (side and top views) with alternating dimers colored differently. (b) McsB is a self-compartmentalized kinase, as shown in the half-cut surface representation. The location of the active sites within the phosphorylation chamber is highlighted in lilac and the pArg194 clamp in orange. The right panel illustrates the high conservation of the respective active site and interface regions. (c) Orthogonal view into the octamer (cross section indicated), with the pArg194 residue highlighted in orange. The right panel illustrates the binding of pArg194 in a complementary charged pocket of the neighboring subunit (top) and its Fo-Fc omit density calculated at 2.5 Å resolution and contoured at 3.0 σ (bottom). (d) Structural details of pArg194 locked by intermolecular contacts to the pR-RS of a neighboring protomer (asterisks indicate residues contributed by the adjacent molecule).

Figure 2—figure supplement 1.. Comparison of McsB_BS and McsB_GS.

(a) Structural alignment of the functional dimer and (b) the pArg binding pocket. The two orthologs are colored differently.

Figure 3.. McsB_BS oligomer conversion depends on a phospho-arginine switch.

(a) Distribution of McsB_BS oligomers at increasing protein concentrations (10–500 nM). (b) Distribution of McsB_GS at increasing protein concentration (10–100 nM). (c) Proposed model of McsB oligomer conversion, with pArg residues represented by colored circles. Structural data are present for dimer and octamer. (d) Mass photometric analysis of the effect of the YwlE phosphatase on McsB_BS oligomer conversion. (e) MS data showing that all pArg residues of autophosphorylated McsB are quantitatively removed, except pArg190 and pArg194. The data show the relative number of phosphoarginine PSMs, plotted as mean ± standard deviation from three technical replicates. For absolute number of PSMs as well as all calculations refer to Source Data File. (f) Mass photometric analysis of the effect of free pArg on McsB_BS oligomer conversion. (g) Mass photometric analysis of the R194K mutant, showing selective destabilization of the octamer. Size markers are indicated.

Figure 3—figure supplement 1.. Shielding of pArg190 and Arg194 in the dimer-dimer interface.

The surface plot shows a half-cut particle (semi-transparent surface), highlighting the burial of residues 190 and 194 in the octameric cage. Other auto-phosphorylated arginine residues shown in Figure 3e are surface exposed and thus accessible to YwlE.

Figure 3—figure supplement 2.. pArg-binding-deficient mutant R337A/D338A.

Size exclusion chromatography analysis of the pArg-binding-deficient mutant R337A/D338A that mainly exists as a dimer. Size markers are indicated.

Figure 4.. McsB_BS dimer and octamer exhibit distinct kinase activities.

(a) Outline of preparing dimeric and octameric McsB_BS (left panel). The established procedure efficiently reconstituted the two B. subtilis kinase forms, as seen in the SEC profiles of the separated McsB_BS samples (right panel). (b) Radiometric ³²P kinase assays visualizing the activity of dimeric and octameric McsB_BS (1 µM) against β-casein (55 µM). ***p≤ 0.001; two-tailed unpaired t test. Data are plotted as mean ± SD (n=3, independent experiments) (c) Radiometric kinase assays using an McsB dilution series. To account for the different enzyme amounts (1000–50 nM), assays were incubated for different times (10–200 min).

Figure 5.. A switch in oligomeric state causes a switch in substrate selectivity.

(a) Radiometric assays of dimeric and octameric McsB_BS (1 µM) using different model substrates. Quantification of activities of dimer (black) and octamer (orange) after 60 min of incubation highlights the distinct substrate preferences (top, representative gel; bottom, quantification of ³²P signal after 60 min, raw data in source data file). Data are plotted as mean ± SD (n=4, independent reactions). Relative activity as mean ± SD. (b) (Top) Kinase assay using the model substrate UNC-45 confirms the substrate filtering role of the octameric cage (top, representative gel; bottom, quantification of ³²P signal after 60 min, raw data in source data file). Data are plotted as mean ± SD (n=4, independent reactions). Relative activity as mean ± SD. (c) Secondary SEC runs of the McsB_BS R190A/R194A mutant enriched in either HOOs (higher-order oligomers) or dimers at 20 µM concentration (d) Radiometric kinase assay of wildtype McsB_BS and the McsB_BS R190A/R194A mutant enriched in either HOOs (higher order oligomers) or dimers (1 µM) against the folded model protein UNC-45_core (top, representative gel; bottom, quantification of ³²P signal after 60 min). Data are plotted as mean ± SD (n=3, independent reactions). Relative activity as mean ± SD.

Figure 5—figure supplement 1.. Quantitative western blot assay of McsB in B.subtilis.

(a) Anti-McsB western blot of heat shocked B. subtilis (left) and of defined amounts of recombinant McsB from B. subtilis (right) used for creating a standard curve. Corresponding loading controls are shown below. (b) Standard curve used for estimating McsB levels in vivo (OD₆₀₀ corrected samples).

Figure 6.. Octamer formation of McsB is critical for the heat-shock response.

(a) In vivo analysis of the R194K McsB oligomerization mutant. Compared to wildtype B. subtilis cells, ∆mcsB and R194K mutant cells exhibit a strong thermo-sensitive phenotype. ****p ≤ 0.0001; one-way ANOVA and Tukey’s multiple comparison test of heat-shocked samples. Data are plotted as mean ± SD (n=5, biological independent samples). Error bars indicate SD. (b) Proposed model illustrating how McsB protein concentration and YwlE activity shape the distribution of McsB oligomers. pArg phospho marks are indicated by red spheres. As shown below, monomer and dimer are prevalent under non-stress conditions. Presumably, their activity on folded proteins (f) can be reversed by the arginine phosphatase YwlE. Under heat-shock, McsB and other components of the stress response machinery are strongly enriched, favoring formation of octameric particles that selectively target misfolded (uf) proteins.

Figure 6—figure supplement 1.. Comparing wildtype and R194K mutant McsB.

(a) Radiometric kinase assay comparing wildtype and R194K mutant McsB (1 µM) against the folded model substrate UNC45_core (top). Fold change in activity of R194K mutant at 30- and 60 min relative to mean of wildtype McsB activity (bottom). **p≤ 0.01; *p≤ 0.05; multiple t test. Data are plotted as mean ± SD (n=3, independent reactions). (b) SEC analysis of autophosphorylated dimeric wildtype and R194K McsB (2.5 µM).

Figure 6—figure supplement 2.. Thermosensitive phenotype of ΔywlE B.subtilis.

(a) In vivo heat shock assay comparing wildtype B. subtilis and the ΔywlE mutant. ****p ≤ 0.0001; one-way ANOVA and Tukey’s multiple comparison test of heat-shocked samples. Data are plotted as mean ± SD (n=3, biological independent samples). Each sample was plated two times (technical replicates) resulting in six data points per column. Error bars indicate SD. (b) Anti-McsB western blot analysis of lysate of heat-shocked wildtype and ΔywlE B. subtilis cultures separated with size exclusion chromatography (bottom) compared to SDS-PAGE of isolated dimeric and higher order McsB (top) serving as size markers.

Author response image 1.. Negative-stain EM of recombinant McsB enriched in HOOs (higher-order oligomers).

Bar; 200 nM. Staining was performed with 2% uranyl acetate. Measured on a FEI Morgagni 268D microscope operated at 80 kV.

Author response image 2.. Anti-mcsB Western blot of wildtype and δ-mcsB B. subtilis at different heat-shock durations.

Red arrows indicate the unspecific 55 and 35 kDa bands.