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. 2013 Jul;41(13):6750-60.
doi: 10.1093/nar/gkt357. Epub 2013 May 9.

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

Stephen P Hancock  1 , Tahereh Ghane, Duilio Cascio, Remo Rohs, Rosa Di Felice, Reid C Johnson
Affiliations

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

Stephen P Hancock et al. Nucleic Acids Res. 2013 Jul.

Abstract

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis-DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis-DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.

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Figures

Figure 1.
Figure 1.
DNA groove widths in the Fis–DNA complex. (A) Crystal structure of Fis bound to the high-affinity binding site F1 (PDB ID: 3IV5). Fis binds DNA as a homodimer (green and purple subunits) and inserts two recognition α-helices (αD) into consecutive major grooves of the target DNA site. The Arg85 side chain, which mediates the only important base contact to the conserved guanines near the outer edges of the binding site, is highlighted in cyan. (B) Major (magenta) and minor (blue) groove widths are plotted over the length of the Fis-bound DNA. Values represent the distance between closest interstrand phosphates minus their van der Waals radii. Dashed lines represent canonical groove widths for B-DNA. The sequence of the F1 site is on the bottom and is color-coded in-line with A. (C) Chemical structures of DNA base pairs used in this study. (D) Space-filling view looking into the minor groove at the center of the F28 (GCG) sequence (blue: N2 atoms, red: O2 atoms). Top is DNA constructed in silico using mean free-DNA parameters (minor groove width at center = 7.1 Å); bottom is the DNA within the Fis complex (minor groove width at center = 4.4 Å).
Figure 2.
Figure 2.
Sequences and Fis-binding properties of DNA duplexes used in this study. (A) Fis-binding isotherms to the different DNA substrates: F1 (black), F28 (blue), F28–dI (green), F28-2AP (red), F29 (magenta) and F29–dI (orange). (B) Sequences and summary of Fis-binding properties of the DNA substrates. I designates inosine, and 2 designates 2-amino purine (2-AP). Fold-worse is relative to F1.
Figure 3.
Figure 3.
Minor groove widths within crystal structures of Fis–DNA complexes correlate with the presence of the purine 2-amino group. (A) Structures of Fis complexes with F28 DNA (blue), F28–dI DNA (green) and F1 DNA (black). Structures are aligned over the proteins, but only the protein of the F28–dI complex is shown. (B) Minor groove width plots of the DNA structures in (A) plus the F28–2AP (red) complex. (C) Structures of Fis complexes with F29 DNA (magenta, PDB ID: 3JRC), F29–dI DNA (orange) and F1 DNA (black). Structures are aligned over the proteins, but only the protein of the F29–dI complex is shown. (D) Minor groove width plots of the DNA structures in (C).
Figure 4.
Figure 4.
Minor groove widths from 50 ns MD simulations. (A) Minor groove width plots over the centers of the F1, F28, F28–dI, F29 and F29–dI sequences. The most probable values along the last 40 ns of the 50 ns MD trajectories are reported (see also Supplementary Figure S3). (B) Mean and minimum values for the most probable minor groove widths over the central 5 bp of the simulated sequences.
Figure 5.
Figure 5.
Neutralization of phosphates by Lys90 across the central narrow minor groove facilitates Fis binding depending on the DNA target sequence. (A) Structure of the Fis–F1 DNA complex highlighting the side chains of Lys90 (blue) and Thr87 (cyan) that contact the DNA phosphate backbone across the narrowest part of the central minor groove. The separation between Thr87 Cα atoms in the Fis dimer is only 20.1 Å. (B) Binding properties of wild-type Fis (wt) and Fis–K90A on different DNA substrates.
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