Competitive binding of MatP and topoisomerase IV to the MukB hinge domain
- Department of Biochemistry, University of Oxford, United Kingdom
- Physical and Theoretical Chemistry Laboratory, University of Oxford, United Kingdom
- The Kavli Institute for Nanoscience Discovery, United Kingdom
Figures
Figure 1 with 1 supplement
ParC dimers and MatP dimers each bind the dimeric MukB hinge with a 1:1 stoichiometry.
(A) Schematics of the basic units of MukBEF complexes, topoIV and a MatP2-matS complex. (B) Example isothermal titration calorimetry (ITC) raw thermogram and binding isotherm following titration of …
Figure 1—figure supplement 1
MatP dimers interact with the MukB hinge domain.
(A) Schematic of the functional domains of MukB and hinge-based truncations (long hinge [LH] and short hinge [SH]). (B) Native mass spectrometry (nMS) confirming that LH is a stable dimer. (C) …
Figure 2 with 1 supplement
The MukB hinge does not form ternary complexes with MatP dimers and topoIV.
(A) Analytical size-exclusion chromatography (SEC). Long hinge (LH), MatP, and ParCCTD were co-incubated at a 1:2:2 ratio at µM concentrations for 1 h prior to injection and separated on a Superose …
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Figure 2—source data 1
Raw data and uncropped gels for Figure 2A.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig2-data1-v2.zip
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Figure 2—source data 2
Raw data for analytical size-exclusion chromatography of Figure 2A.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
The MukB hinge does not form ternary complexes with MatP-matS complexes and topoIV.
(A) Representative mass spectra indicating that 50 bp matS-containing DNA specifically binds MatP dimers under the conditions used. MatP and DNA was mixed at 1:1. Gray italics denote the theoretical …
Figure 3 with 1 supplement
MatP and ParC compete for binding to overlapping sites on the MukB hinge.
(A) Fluorescence correlation spectroscopy (FCS) measurements of competition for binding between ParC and MatP for MukB and also between MatP and 15 bp DNA hairpins, containing a 13 bp matS2 site or …
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Figure 3—source data 1
Raw data and uncropped gel for Figure 3B.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-data1-v2.zip
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Figure 3—source data 2
Raw data and uncropped gels for Figure 3C.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-data2-v2.zip
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Figure 3—source data 3
Raw data for analytical size-exclusion chromatography of Figure 3C.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-data3-v2.xlsx
Figure 3—figure supplement 1
MatP and ParC compete for binding to overlapping sites on the MukB hinge.
(A) Autocorrelation curves of Cy3B-MatP (1 nM, red), TAMRA-MukBS718TAG (1 nM, blue), and a mixture of Cy3B-MatP (1 nM) and unlabeled MukB (400 nM, green). Measured diffusion times of Cy3B-MatP, …
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Figure 3—figure supplement 1—source data 1
Raw and fitted data for autocorrelation curves in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Raw data for triplicate ATPase assay in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-figsupp1-data2-v2.xlsx
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Figure 3—figure supplement 1—source data 3
Tabulated data including fitted data for Figure 3—figure supplement 1D.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-figsupp1-data3-v2.xlsx
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Figure 3—figure supplement 1—source data 4
Raw data of that presented in Figure 3—figure supplement 1E.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig3-figsupp1-data4-v2.xlsx
Figure 4 with 1 supplement
matS sites compete with the hinge for MatP binding.
(A) Representative mass spectra of species detected between long hinge (LH) and MatP in the presence of matS DNA. LH, MatP, and in the case of the upper panel matS, were mixed at 1:2:1. Gray italics …
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Figure 4—source data 1
Raw data and uncropped gels for Figure 4B.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig4-data1-v2.zip
Figure 4—figure supplement 1
matS sites compete with the MukB hinge for MatP binding.
(A) Native PAGE analysis of impaired long hinge (LH) and matS/non-specific DNA binding by MatP4A (K71A, Q72A, R75A, and R77A) and MatP4E (K71E, Q72E, R75E, and R77E). Gels were stained with …
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Figure 4—figure supplement 1—source data 1
Raw data and uncropped gels for Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/70444/elife-70444-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
Cells expressing MukBKKK are impaired in ParC binding and exhibit defects in MukBEF function.
(A) MukBEF phenotype of MukB+, MukBKKK, and ΔmukB cells as judged by temperature-sensitive growth in rich medium (LB) at 22°C and 37°C. Basal levels of plasmid-borne MukB and MukBKKK were expressed …
Figure 5—figure supplement 1
MukBEF distances to ori1/ter3 and marker localizations.
(A) Box plots of median cell length of data acquired for Figure 5B. Wild-type (WT) MukB, 4837 cells, median cell length 2.87 μm; MukBKKK, 5846 cells, median cell length 2.98 μm; ΔmukB, 10,670 cells, …
Tables
Table 1
Theoretical masses of proteins and DNA used in native mass spectrometry (nMS).
Predicted and measured masses of MukBEF, topoIV, and MatP components or variants and also DNA substrates. Errors are the standard deviation in mass determination.
| Protein | Theoretical mass of monomer (Da) * | Measured oligomeric state(s) | Theoretical mass(es) of native state(s) (Da) | Measured mass(es) (Da) |
|---|---|---|---|---|
| LH (MBP cleaved) | 34334.87 | Dimer | 68669.74 | 68668 ± 1 |
| SH-His | 15204.73 | Monomer/dimer mix † | 15204.73/30409.46 | 15105 ± 530188 ± 12 |
| MatP(i.e., MatPΔ18C-His) | 18329.85 | Dimer | 36659.7 | 36418 ± 1 |
| MatP K71E, Q72E, R75E, R77E | 18277.63 | Dimer | 36555.26 | 36429 ± 15 |
| His-ParC | 86152.75 | Dimer | 172305.5 | 172076 ± 2 |
| His-ParC R705E R729A | 86040.57 | Dimer | 172081.14 | 171863 ± 3 |
| MBP-ParCCTD | 73422.74 | Monomer | 73422.74 | 70761 ± 6‡71447 ± 8‡ |
| ParE-His | 72484.36 | Dimer | 144968.72 | 144717 ± 2 |
| 50 bp matS DNA | 30925.10 | N/A | 30766.82 | 30801 ± 14 |
| 50 bp nonspecific DNA | 30925.10 | N/A | 30766.82 | 30780 ± 15 |
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*
Masses include first methionine.
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†
From this work.
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‡
Degradation products.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70444/elife-70444-transrepform1-v2.docx
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Source code 1
All custom MATLAB scripts used for analysis of epifluorescence microscopy data in Figure 5 and Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/70444/elife-70444-supp1-v2.zip
