Single-domain antibody inhibitors target the coiled coil arms of the Bacillus subtilis SMC complex
- Department of Fundamental Microbiology (DMF), Faculty of Biology and Medicine (FBM), University of Lausanne (UNIL), Switzerland
- Institute of Medical Microbiology, University of Zurich, Switzerland
Figures
Figure 1 with 2 supplements
Selection of sybodies targeting Smc-ScpAB in B. subtilis.
(A) (i) Structural design and randomization scheme of the three synthetic sybody libraries: concave, loop, and convex. Complementarity-determining regions (CDRs) 1, 2, and 3 are shown in yellow, orange, and red, respectively; randomized residues are shown in stick representation. Adapted from Zimmermann et al., 2018. (ii) SMC domain organization. Hd: head, Hng: hinge, 4N: 4N arm-to-arm contact, J: joint. (iii) SMC complexes harbor juxtaposed arms in their ‘closed’ state (left panel). Upon ATP binding at the heads, the heads engage, leading to an ‘open’ state in which the arms disengage (right panel). This state is thought to accomodate a segment of DNA (not shown) in the S compartment formed by the SMC arms, that will be pushed towards the K compartment formed by the heads and kleisin, after ADP release. Blue: SMC dimer, Green: kleisin ScpA. The ScpB KITE subunits are not represented for the sake of simplicity. HngDBS: Hinge DNA-binding site, HdDBS: Head DNA-binding site. (B) Framework for sybody selection. In vitro selection starts with ~1012 sybody variants per library subjected to ribosome display for pre-enrichment, followed by two rounds of phage display. For in vivo selection, 95 randomly selected sybody genes were integrated into the B. subtilis chromosome under the control of a xylose-inducible promoter by allelic replacement at the amyE locus. Growth defects on rich medium were tested on ONA agar plates with or without 0.5% xylose. Example shown: Sb164 (loop library) did not affect growth, whereas Nb31 impaired growth upon induction, suggesting interference with bsuSmc function. (C) Transformation assay results for a B. subtilis straintransformed with one out of 95 sybodies of the loop library expressed from a xylose-inducible promoter . Bars show colony counts ‘without’ (green) on top of ‘with’ (pink) xylose. Strains are ordered by their original position in the 96-well plate. Fourteen sybodies consistently impaired colony formation under inducing conditions (marked by dotted lines). Sybody numbers indicated above the plots correspond to selected candidates used in subsequent experiments, numbering according to order of first use. Notably, the E09 sybody (Sb018) also showed an absence of transformants upon sybody induction. However, this sybody candidate gave intermediate phenotypes in later experiments, which is why it was excluded from detailed analysis.
Figure 1—figure supplement 1
Preparation of bsuSmc(C119S, C437S, C826S, E1118Q, R643C)-ScpAB complex and loop extrusion model.
(A) Size-exclusion chromatography profile of biotinylated bsuSmc(C119S, C437S, C826S, E1118Q, R643C)-ScpAB complex. Peak fractions (elution at ~13 mL, pooled fractions indicated by dotted lines for a final volume of 1.8 mL) were collected for downstream use; the final concentration was 5.9 µM (dimer). *A secondary peak appeared at ~14 mL; its identity was uncertain; it was excluded from the pooled fraction. Pulldown experiments confirm successful biotinylation of the bsuSmc(C119S, C437S, C826S, E1118Q, R643C)-ScpAB complex. (B) Segment-capture model for DNA translocation by bsuSmc-ScpAB, in which ATP binding and hydrolysis drive transitions between open and closed conformations to mediate loop extrusion.
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Figure 1—figure supplement 1—source data 1
Original TIF image file of protein gel shown in Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/111131/elife-111131-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing image of protein gel shown in panel Figure 1—figure supplement 1A with lanes and bands labeled.
- https://cdn.elifesciences.org/articles/111131/elife-111131-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Representative results from in vivo sybody selection based on colony formation.
(A) Representative examples of B. subtilis transformation assay for nine sybodies . Sybody genes were integrated into the amyE locus under xylose-inducible control. Transformants were grown on oxoid nutrient agar (ONA) plates supplemented with chloramphenicol and 0.5% xylose to assess growth defects. Shown are candidates from the concave library: Sb051 (well B08) and Sb078 (D12). Loop library: Sb007 (E05), Sb020 (E12), Sb031 (F05), Sb164 (D12), and NbH07 (Sb194). Convex library: Sb239 (D12) and Sb248 (E10). (B) Transformation assay results for the 285 B. subtilis strains expressing individual xylose-inducible sybodies. Bars show each colony counts ‘without’ (green) on top of ‘with’ (pink) xylose. Strains are ordered by their original 96-well plate. Only the loop library yielded sybodies that consistently impaired growth under inducing conditions (dotted line). Sybody numbers indicated above the plots correspond to positive candidates used in subsequent experiments, numbered according to order of use. Results for the loop library are also shown in Figure 1.
Figure 2 with 3 supplements
Sybody-induced chromosome segregation defects visualized by ParB-GFP imaging.
(A) (i) Top: schematic illustrating ParB-GFP binding near oriC, enabling visualization of origin positioning. Bottom: representative images showing ParB-GFP foci in wild-type (‘WT’) and Δsmc B. subtilis. WT cells typically display 2–4 foci per cell, whereas Δsmc cells exhibit reduced foci numbers. (ii) Number of ParB-GFP foci per µm of cell length in a strain carrying inducible sybody Sb006. A significant decrease in foci is detected from 40 minutes post-induction (pₜ₀₋₁₀=0.9674; pₜ₀₋₂₀=0.9033; pₜ₀₋₃₀=0.3744; pₜ₀₋₄₀=0.0145; pₜ₀₋₅₀=0.0004; pₜ₀₋₆₀<0.001). Based on this, a standardized induction time of ~35 minutes was used in subsequent experiments. (B) ParB-GFP foci density (foci/µm) in WT, Δsmc, and sybody-expressing strains after 35 minutes of xylose induction. Violin plots show distribution per condition; solid lines denote the mean, and dotted lines indicate quartiles. Several hundreds of cells were analyzed (between 351 and 1735), except for Δsmc and Sb010 were fewer cells were available (134 and 73, respectively). (C) Spot assay to assess colony formation of B. subtilis strains harboring bsu or spn variants of Smc-ScpAB and ParB. Top: Schematic shows gene origin (blue: B. subtilis; gray: S. pneumoniae). The leftmost column corresponds to the parental B. subtilis strains without vector integration lacking chloramphenicol (‘Cm’) resistance (non-growing). The next spots represent the same strains carrying the Cm resistance but lakcing a sybody gene (EV for empty vector). Remaining columns are sybody-expressing strains; sybody numbers are indicated . Cells were grown for 16 hours at 37°C on ONA supplemented with 0.5% xylose and chloramphenicol. Hd: head, Hng: hinge, 4N: 4N arm-to-arm contact, J: joint.
Figure 2—figure supplement 1
Functional impact of sybody expression on chromosome organization, cell length, and growth in B. subtilis.
(A) Average number of ParB-GFP foci per µm of cell length in strains expressing individual sybodies, with and without xylose induction. Most uninduced strains differed significantly from the Smc WT control, likely due to leaky expression from the Pxyl promoter (p<0.0001), except Sb015 (p>0.9999), Sb156 (p=0.2345), and Sb016 (negative control, p>0.9999). (B) Average cell length across the same conditions, measured in ParB-GFP strains with or without Smc, and with or without sybody. Bars indicate standard deviation. Tested sybodies caused cell elongation, a characteristic phenotype of impaired Smc activity, as chromosome segregation defects delay cell division, with mean lengths from 4.70±2.16 µm (Sb020) to 8.68±2.99 µm (Sb010), compared to 4.51±1.95 µm in wild-type and 9.96±8.92 µm in Δsmc. (C) Growth curves of B. subtilis strains expressing individual sybodies compared to the WT strain, with and without xylose induction. Each curve represents the mean of two biological replicates. Six sybodies were randomly picked and shown in this figure. Strains showed wild type-like growth in the absence of xylose but strong delays post-induction, followed by partial growth recovery after 12 hours, likely due to xylose depletion or suppressor emergence. In sybody-expressing strains, a drop in cell density was observed ~2.5 hours post-induction.
Figure 2—figure supplement 2
Sybody-GFP expression at different inducer concentrations.
Selected Sybody-GFP proteins (ΔamyE::sybody-mGFP::CamR::amyE) were tested by GFP imaging at different inducer concentrations (0, 0.005, 0.05 and 0.5% xylose). Representative images are shown. Notably, sybody Sb007 (E5, loop library) generates a mild growth phenotype (smaller colonies but normal colony numbers, Figure 1A and B) but shows good expression levels and focal localization (top row).
Figure 2—figure supplement 3
Imaging of various Sybody-GFP proteins without inducer.
Selected Sybody-GFP constructs (ΔamyE::sybody-mGFP::CamR::amyE) were grown in the absence of inducer and tested for gfp expression. Representative images are shown.
Figure 3 with 1 supplement
Mapping sybody binding sites on Smc-ScpAB.
(A) Colony formation of B. subtilis strains harboring chimeric Smc proteins comprising S. pneumoniae and B. subtilis sequences and expressing sybodies The schematic above the spot assay depicts the species origin of various parts of the smc gene (blue: B. subtilis; gray: S. pneumoniae). ‘–’ indicates no insertion at the amyE locus; ‘EV’ refers to the empty vector control containing only the chloramphenicol resistance cassette at amyE; numbered labels correspond to sybodies. Cells were spotted on nutrient rich medium (ONA) supplemented with chloramphenicol and xylose and incubated for 16 hours at 37°C. Hd: head, Hng: hinge, 4N: 4N arm-to-arm contact, J: joint. (B) ATP hydrolysis rates of bsuSmc-ScpAB in the presence of sybodies but absence of DNA. Means and standard deviation from three technical replicates are indicated. Individual datapoints are shown. Significant effects by one-way ANOVA are indicated by p values. (C) ATPase rates in the presence of 40 bp dsDNA. All sybodies reduced DNA-stimulated ATP hydrolysis. Means and standard deviation from four technical replicates are indicated. Individual datapoints are shown. Reported p-values: Sb020 (p=0.340), Sb194 (p=0.0049), Sb002 (p=0.0007), Sb015 (p=0.0003); all others, p<0.0001. (D) Schematic summary of sybody binding sites mapped onto the Smc dimer, categorized by their effect on ATPase activity. Sybodies are grouped based on functional impact and mapped to corresponding structural regions: pink/yellow boxes indicate residues 318–339 and 836–857; green boxes mark residues 248–276 and 899–927; and the red box highlights the 4N contact region (approx. residues 290–320).
Figure 3—figure supplement 1
Sybody effects on cellular viability and SMC ATPase rate.
(A) Colony formation assay assessing the growth of B. subtilis strains carrying chimeric Smc-ScpAB genes composed of S. pneumoniae and B. subtilis components. Strains harbor individual sybody constructs integrated at the amyE locus were grown without inducer. ‘–’ indicates no integration at amyE; ‘EV’ corresponds to an empty vector control containing only the chloramphenicol resistance cassette. Cells were spotted on nutrient rich medium (ONA) containing chloramphenicol and incubated for 16 hours at 37°C. (B) Overall ATPase hydrolysis rate of the bsuSmc-ScpAB complex in presence of each sybody and +/- DNA. Means and standard deviation from four technical replicates are indicated. Individual datapoints are shown. Sb06, 021, and 194 triggered a significant difference in ATP hydrolysis between +/- dsDNA conditions. Other results are non-significant.
Figure 4 with 1 supplement
Proposed models for sybody interactions with bsuSmc-ScpAB .
Model for sybodies of the OpeningUP, ClosingUP, and ClosingDOWN group. OpeningUP sybodies likely prevent complete arm closure. In the presence of DNA, these sybodies may stabilize an ‘open’ conformation and impede head disengagement, resulting in reduced ATPase activity (not shown here). ClosingUP and ClosingDOWN sybodies stabilize a closed-arm conformation. These potentially hinder DNA segments from entering the inter-arm space and accessing the hinge-proximal DNA binding site.
Figure 4—figure supplement 1
Hypothetical model for an OpeningDOWN class of sybodies, not recovered in this study.
This may indicate that stabilizing an open conformation near the ATPase heads does not interfere with Smc function.
Tables
Table 1
Sybody enrichments at different steps of the selection procedure.
Ribosome display output was quantified by qPCR, while phage display results include final phage titers and enrichment values from rounds one and two, also measured by qPCR.
| Ribosome display | Phage display | |||
|---|---|---|---|---|
| (qPCR) | (qPCR) | |||
| Library | Total # of RNAs | Titer (PFU/mL) | Enrichment 1 | Enrichment 2 |
| Convex (S) | 1.36×108 | 8.96×1013 | 3.1× | 1474.7× |
| Loop (M) | 6.95×107 | 5.46×1013 | 1.4× | 1209.9× |
| Concave (L) | 1.25×108 | 8.32×1013 | 1.8× | 1808.0× |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | E. coli SS320 | Lucigen | Cat# 60512-1 | For phage display |
| Strain, strain background (E. coli) | E. coli MC1061 | Lucigen | Cat# 60514-1 | Protein production |
| Strain, strain background (E. coli) | E. coli BL21-Gold (DE3) | Novagen | Cat# 69450-3 | Protein production |
| Strain, strain background (Bacillus subtilis) | B. subtilis strain 168 | Bacillus Genetic Stock Center | 1A700 | BSG1001, trpC2 |
| Recombinant DNA reagent | M13KO7 helper phage | New England Biolabs | Cat# N0315S | For phage production |
| Recombinant DNA reagent | Plasmid pSBinit | AddGene | RRID:Addgene_110100 | Vector for initial sybody expression |
| Recombinant DNA reagent | Plasmid pET-28a(+) | AddGene | RRID:Addgene_141289 | Vector for protein expression |
| Peptide, recombinant protein | Neutravidin | Thermo Scientific | Cat# 31000 | For plate coating |
| Commercial assay or kit | Dynabeads MyOne Streptavidin T1 | Invitrogen | Cat# 65601 | Target immobilization |
| Commercial assay or kit | Dynabeads MyOne Streptavidin C1 | Invitrogen | Cat# 65001 | Target immobilization |
| Commercial assay or kit | HiTrap Blue HP 5 mL | Cytiva | Cat# 17041301 | Affinity chromatography |
| Commercial assay or kit | HiTrap Heparin HP 5 mL | Cytiva | Cat# 17040701 | Affinity chromatography |
| Commercial assay or kit | HiTrap Butyl HP 5 mL | Cytiva | Cat# 28411005 | Hydrophobic interaction chromatography |
| Commercial assay or kit | HiTrap Q HP 5 mL | Cytiva | Cat# 17115401 | Ion exchange chromatography |
| Commercial assay or kit | Superose 6 10/300 Increase | Cytiva | Cat# 29091596 | Size-exclusion chromatography |
| Commercial assay or kit | Superose 6 PG XK 16/70 | Cytiva | Cat# 90100042 | Size-exclusion chromatography |
| Commercial assay or kit | HiLoad Superdex 75 PG 16/600 | Cytiva | Cat# 28-9893-33 | Size-exclusion chromatography |
| Commercial assay or kit | Sepax SRT-10C SEC100 c | Sepax Technologies | Cat# 239100-10030 | Size-exclusion chromatography |
| Chemical compound, drug | EZ-Link Maleimide-PEG2-Biotin | Thermo Scientific | Cat# A39261 | Protein biotinylation |
| Chemical compound, drug | Tris(2-carboxyethyl) Phosphine Hydrochloride (TCEP) | Sigma-Aldrich | Cat# 646547 | Reducing agent |
| Commercial assay or kit | Zeba spin 7K MWCO 0.5 mL | Thermo Scientific | Cat# 89882 | Protein desalting spin columns |
| Commercial assay or kit | Nunc Maxisorp 96-well immunoplates | Merck | Cat# M9410 | Immobilization of sybodies for ELISA |
| Peptide, recombinant protein | Protein A from S. aureus | Merck | Cat# P3838 | Immobilization of sybodies for ELISA |
| Antibody | Mouse monoclonal anti-myc | Sigma-Aldrich | Cat# M4439; RRID:AB_439694 | Immobilization of sybodies for ELISA; 100 µL of a 1:2000 dilution |
| Commercial assay or kit | His MultiTrap HP | Cytiva | Cat# 28400989 | Affinity chromatography |
| Peptide, recombinant protein | Pyruvate Kinase/Lactate Dehydrogenase | Sigma-Aldrich | Cat# P0294-5ML | Enzyme-coupled ATPase measurement |
| Chemical compound, drug | Phosphoenol-pyruvic acid | Sigma-Aldrich | Cat# P7002-100MG | Enzyme substrate |
| Chemical compound, drug | Nicotinamide adenine dinucleotide hydrate (NADH) | Santa Cruz | Cat# 205762A | Enzyme substrate |
| Software | GraphPad Prism | GraphPad | RRID:SCR_002798 | Scientific graphing and curve fitting |
Additional files
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Supplementary file 1
ELISA sybody binding assay.
- https://cdn.elifesciences.org/articles/111131/elife-111131-supp1-v1.xlsx
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Supplementary file 2
Sybody sequences.
- https://cdn.elifesciences.org/articles/111131/elife-111131-supp2-v1.xlsx
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Supplementary file 3
Percentage of cells lacking ParB-GFP foci, indicating the absence of chromosome due to segregation defect.
In presence and absence of xylose for sybody induction.
- https://cdn.elifesciences.org/articles/111131/elife-111131-supp3-v1.xlsx
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Supplementary file 4
Strains, Plasmids, Oligonucleotides.
- https://cdn.elifesciences.org/articles/111131/elife-111131-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/111131/elife-111131-mdarchecklist1-v1.pdf
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Single-domain antibody inhibitors target the coiled coil arms of the Bacillus subtilis SMC complex
eLife 15:RP111131.
https://doi.org/10.7554/eLife.111131.3
