Figure 1.
DNA-binding domain of selected members of the H-NS family of proteins and DNA fragment optimization.
(A) Sequence alignment of the C-terminal domain of the following proteins: Ler; chromosomal H-NS of E. coli (ecHNS); Shigella flexneri (sfHNS); Salmonella enterica serovar Typhimurium (seHNS); Yersinia enterocolitica (yeHNS); the plasmid R27-encoded H-NS protein (pR27); and E. coli StpA. The secondary structure elements of DNA-bound CT-Ler and free H-NS are shown. Highly conserved residues within the consensus DNA-binding motif are highlighted in red. (B) Analysis of the interaction of CT-Ler with 30 bp DNA fragments (LeeA-G, sequences are listed in Table S1) derived from the DNAse I footprint of Ler in the LEE2/LEE3 regulatory region [10]. Complex formation was followed by the increase of CT-Ler fluorescence anisotropy. (C) Fluorescence anisotropy titrations of CT-Ler with LeeH (black circle) and LeeFG (gray circle). Solid curves are the best fit to a model assuming a 1∶1 complex. The point by point deviations between fitting and experimental points are shown in the top panel.
Figure 2.
NMR analysis of the CT-Ler/LeeH interaction.
(A) Mean absolute changes in 1H-NMR chemical shifts caused by the addition of 0.5 equivalents of CT-Ler. The average is over all resolved resonances per nucleotide. The upper and lower LeeH strands are identified by black and gray bars, respectively. (B) Backbone amide chemical shift changes in CT-Ler () upon complex formation with LeeH. The scaling factor
corresponds to the ratio of 15N and 1H magnetogyric constants. Resonances that were not observed are denoted by # (Gly87) or * (Pro92).
Figure 3.
Structure determination of the CT-Ler/LeeH complex based on NMR and SAXS.
(A) SAXS intensity in logarithmic scale measured for a CT-Ler/LeeH equimolar sample (open circles) as a function of the momentum transfer , where
Å is the X-ray wavelength and
is the scattering angle. CRYSOL fit of the SAXS curve using a representative NMR structure (red); the average deviation
is 1.16. Only the range 0.018< s <0.4 Å−1 is displayed. The point by point deviations [(I(s)exp−I(s)fit)/
], where
is the experimental error are shown in the bottom panel. (B) Scatter plot of NMR intermolecular restraint violations versus
values for the initial set of 400 complex structures and the final ensemble of 20 low energy structures highlighted in red (inset). The main panel shows a zoom of the best structures. (C) Backbone overlap of the 20 lowest energy complex structures. Protein backbone is coloured in rainbow gradation.
Figure 4.
(A) Structure of CT-Ler/LeeH complex. CT-Ler is shown as a ribbon diagram and transparent surface representation. Interactions involve the DNA minor groove and Loop2 and the α–helix of CT-Ler. (B) Close-up view of the binding interface. CT-Ler residues involved in DNA recognition are shown as stick models. The electrostatic potential of LeeH, calculated with DelPhi in the absence of CT-Ler, is shown. (C) Electrostatic potential of CT-Ler. The orientation of the complex is the same as in A. (D) Schematic representation of the hydrophobic (dashed lines) and polar (solid lines) intermolecular contacts.
Figure 5.
DNA recognition by CT-Ler is dictated by the minor groove width.
(A) Stick representation of Arg90 side chain inserted at the floor of the negatively charged LeeH minor groove. The electrostatic potential of LeeH, calculated in the absence of CT-Ler, is plotted on the LeeH surface. (B) Average minor-groove width (blue) and electrostatic potential in the centre of LeeH minor groove (red). The position of the guanidium group of Arg90 is indicated. (C-D) Helical parameters of LeeH in complex with CT-Ler. Roll and helix twist angles are shown. Dashed lines correspond to values typical of canonical B-DNA [56].
Figure 6.
Arg90 is essential for DNA-binding.
(A) Fluorescence anisotropy titrations of wild type, R90K, R90Q and R90G CT-Ler with LeeH. (B) EMSA of wild type and mutant Ler proteins. 80 ng of DNA (LEE2 positions −225 to +121) were incubated with the indicated Ler concentrations and analyzed on a 1.5% agarose gel. 1 Kb DNA ladder was included as a reference (lane M).
Figure 7.
Minor groove shape serves as a signature for CT-Ler/DNA recognition.
(A) CT-Ler binding to DNA variants containing single base-pair substitutions with respect to LeeH (wt). The LeeH minor groove width is also shown to highlight the fact that mutations in the compressed and expanded regions of the minor groove caused the largest effects. (B) Relative Kd values of the complexes formed between CT-Ler and 10-mer duplexes with different AT-rich sequences. The most stable complex, used as reference, has the AATT sequence present in LeeH. Relative Kd values are Kd(mutant)/Kd(reference) determined by fluorescence anisotropy.
Figure 8.
The DNA-binding domains of Ler and H-NS share a similar indirect DNA readout mechanism.
(A) EMSA (1.5% agarose) of the −225 to +121 LEE2 fragment (80 ng) with increasing concentrations of wild type and R114G H-NS proteins. (B) DNA titrations of CT-H-NS followed by NMR. Expansions of 1H-15N HSQC spectra of CT-H-NS in the presence of the 10 bp duplexes AATT (top left, 0, 0.5, 1, 2, 3 and 4.5 equivalents) or TATA (top right, 0, 1, 2, 3, 4.5 and 6 equivalents). The DNA-dependent shifts of selected cross-peaks were fitted to a 1∶1 model (bottom), supported by the strict linear displacement of the cross-peaks during the titration. (C) CT-Ler and CT-H-NS binding to the −225 to +121 LEE2 fragment (20 ng) followed by EMSA on a 7% polyacrylamide gel.