Mechanism of environmentally driven conformational changes that modulate H-NS DNA-bridging activity
- Leiden University, Netherlands
- Van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam, Netherlands
- University of Heidelberg, Germany
Figures
Figure 1 with 5 supplements
Modulation of H-NS function by ionic conditions.
(a) DNA-bridging efficiency as a function of H-NS concentration in the presence of 10 mM MgCl2. (b) DNA-bridging efficiency as a function of MgCl2 concentration. (c) DNA-bridging efficiency as a …
Figure 1—figure supplement 1
Multimeric state of H-NS measured using size exclusion chromatography.
(a) Elution pattern of 50 and 100 µM of H-NS as a function of Mg2+. (b) Elution pattern of 50 and 100 µM of H-NSY61DM64D as a function of Mg2+. Lines are to guide the eye.
Figure 1—figure supplement 2
Electrophoretic Mobility shift assay.
(a) H-NS and (b) H-NSY61DM64D. The proteins were added to 32P-labeled 685 bp DNA substrate at 0.4, 0.8, 1.2, 1.6, 12.4, 3.3, 6.6 µM concentrations. H-NS and H-NSY61DM64D exhibit similar DNA-binding …
Figure 1—figure supplement 3
DNA recovery of H-NS and.
H-NSY61DM64D in the DNA-bridging assay.
Figure 1—figure supplement 4
Schematic depiction of techniques used in this study.
(a) Schematic depiction of TPM and the effect of increased protein binding to DNA on the Root Mean Squared (RMS) motion of the bead. (b) Schematic depiction of the DNA-bridging assay. …
Figure 1—figure supplement 5
Modulation of H-NS by alternative anions.
(a) Extension of DNA as a function of the K-glutamate concentration (N > 70, for each point). (b) DNA-bridging efficiency as a function of the K-glutamate concentration. Error bars indicate standard …
Figure 2 with 11 supplements
Conformation of the H-NS dimer as a function of osmolarity.
(a) Snapshots depicting representative conformations of H-NS in the simulations with 50 mM KCl (top) and 10 mM MgCl2 +50 mM KCl (bottom) (For the full movies depicting these effects see Figure …
Figure 2—figure supplement 1
Examples of ‘closed’ H-NS conformations in the presence of (a) 50 mM KCl, (b) 130 mM KCl, or (c) Hha.
https://doi.org/10.7554/eLife.27369.010
Figure 2—figure supplement 2
Contact maps of full-length H-NS dimers simulations in different conditions.
(a) 50 mM KCl, (b) 10 mM MgCl2 +50 mM KCl, (c) 130 mM KCl, (d) 10 mM MgCl2 +130 mM KCl, (e) Hha +50 mM KCl, (f) Hha +10 mM MgCl2 +50 mM KCl, Residues are considered in contact if the smallest …
Figure 2—figure supplement 3
Time traces of the O-H distance between residues 45 and 49.
The time traces are shown in different shades of gray to indicate the different runs. The red dashed line indicates the distance threshold for forming a hydrogen bond. The red and green lines are …
Figure 2—figure supplement 4
Location of K+on hr-NS.
The probability of finding K+ ions within 0.6 nm of an H-NS residue, PK+, is plotted as function of the residue index for (a) 50 mM KCl and (b) 130 mM KCl in the presence and absence of MgCl2.
Figure 2—figure supplement 5
Contact maps of H-NS dimers in different conditions, focused on the interactions between the dimerization domain and the DNA-binding domain.
(a) The probability of finding the DNA-binding domain within 0.6 nm of residues in the dimerization domain, P96-137 (b) The probability of finding the dimerization domain within 0.6 nm of residues …
Figure 2—figure supplement 6
Location of Hha on H-NS.
(a) The probability of finding Hha within 0.6 nm of an H-NS residue, PHha, is plotted as a function of the residue index. The PHha is indicated on a surface representation of an H-NS dimer in open …
Figure 2—figure supplement 7
Correlation between hydrogen bond distance and proximity of Mg2+to the buckle in helix α3.
The correlation between the hydrogen bond distance dO45-N49 and the minimum distance between Mg2+ ions and a residue dmin is shown as a contour plot. The darker colors indicate higher probabilities …
Figure 2—figure supplement 8
Mg2+localization on H-NS.
The probability of finding Mg2+ within 0.6 nm of H-NS in the presence of (a) 50 mM KCl, (b) 130 mM KCl, or (c) Hha. This probability is indicated on a surface representation of an H-NS dimer in open …
Figure 2—figure supplement 9
Function of the H-NS derivative, H-NSE43A,E44A,S45A.
(a) DNA-bridging efficiency of H-NSE43A,E44A,S45A as a function of the Mg2+ concentration. (b) Root Mean Square displacement (RMS) of DNA in the presence of H-NSE43A,E44A,S45A.
Figure 2—video 1
Conformational flexibility of H-NS.
The protein is rendered in cartoon representation with a transparent surface. The color code indicates the domain organization of H-NS: blue - site 1 (residue 1–40), red - the buckle region in helix …
Figure 2—video 2
The effect of magnesium on the conformational flexibility of H-NS.
The protein is rendered in cartoon representation with a transparent surface. The color code indicates the domain organization of H-NS: blue - site 1 (residue 1–40), red - the buckle region in helix …
Figure 3 with 2 supplements
Modulation of H-NS function by protein cofactors.
(a) DNA bridging efficiency as a function of inhibiting peptides targeting either the dimerization domain (H-NS1-58) and multimerization domain (H-NS56-82). (b) Root Mean Square displacement (RMS) …
Figure 3—figure supplement 1
McGhee-von Hippel analysis of H-NS DNA binding curves based on TPM data.
Binding curves of (a) H-NS, (b) H-NS + Hha TPM data as a function of Mg2+, and (c) H-NS, H-NS + Hha, H-NS + YdgT, and H-NSE43A,E44A,S45A (d) Fit variables for all datasets, the binding affinity (K), …
Figure 3—figure supplement 2
DNA-bridging efficiency of H-NS (red), H-NS + 4 µM YdgT (blue) and H-NSE43A,E44A,S45A (orange) as a function of MgCl2 concentration.
https://doi.org/10.7554/eLife.27369.023
Figure 4
Model of H-NS complex assembly.
(a) H-NS nucleates at preferred DNA sequences in the genome. (b) H-NS laterally multimerizes laterally along the DNA in the ‘closed’ conformation. (c) In the presence of Mg2+ or other H-NS …
Author response image 1
DNA binding and activity of H-NS and H-NSC21Alexa555.
(A) Electrophoretic mobility shift assay using a 32P labeled (AT-rich) curved DNA substrate as described in Dame et al., Bioch., 2001. (B) The root mean squared (RMS) displacement of a DNA tether …
Author response image 2
DNA binding by the H-NS derivative H-NSR11E measured by the Root Mean Square displacement of DNA.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.27369.025
