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. 2010 Apr 15;24(8):814-26.
doi: 10.1101/gad.1900610.

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

Stefano Stella  1 , Duilio Cascio, Reid C Johnson
Affiliations

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

Stefano Stella et al. Genes Dev. .

Abstract

The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of approximately 65 degrees , and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

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Figures

Figure 1.
Figure 1.
DNA binding by Fis. (A) EMSA showing binding to a 97-bp fragment containing the F1 site in the presence or absence of competitor DNA [50 μg/mL poly(dI • dC)] with fourfold increasing amounts of Fis beginning at 0.005 nM. Fis generates an additional three nonspecifically bound complexes on the same DNA fragment without competitor DNA. (B) Sequence logo qualitatively depicting the degenerate Fis-binding motif based on compilations of native Fis-binding sites, SELEX experiments, and mutagenesis (Finkel and Johnson 1992; Hengen et al. 1997; Ussery et al. 2001; Cho et al. 2008; Shao et al. 2008b). Bases denoted below the numbers are inhibitory for binding. (C) Sequence of the 27-bp optimal F1 and F2 sites used for crystallography and nonconsensus sites F3 (AT center) and F4 (GC center). In this and subsequent figures, sequences where crystal structures of Fis complexes were determined are demarked with an asterisk, and differences from the F1 sequence are in lowercase. Fis-binding affinities (Kd) with standard deviations and dissociation rates (T1/2) for the 27-bp oligonucleotides are shown.
Figure 2.
Figure 2.
Fis–DNA crystal structure and packing. (A) Fis dimer bound to the F1 sequence. Nucleotides where base identity is important for binding are highlighted in yellow. (B) View of the crystal lattice. The DNA from one asymmetric unit is highlighted in orange. (C) Stacking of DNA ends from adjacent asymmetric units. (D) Interactions between opposing Fis dimers stabilizing the lattice. (E) Fis–DNA contacts in the F1 complex. H bonds to the bases and phosphate backbone are denoted on the structure and ladder representations.
Figure 3.
Figure 3.
Structural distortions in the Fis-bound DNA. (A) Roll angle deviations at base-pair steps from the F1 complex structure (orange) and F2 complex structure (blue), as aligned with the sequences on the top. (B) DNA in the F1 structure, with bases important for Fis binding highlighted in green. Red lines schematically represent the DNA helix axis, highlighting the positions of bends. The twist values over the segments denoted in the rectangles are averaged from the F1 structure. (C) Major and minor groove widths for the F1 and F2 structures. Values are between closest phosphates minus their van der Waals surfaces. Dashed lines denote canonical groove widths from DNA fibers (Chandrasekaran and Arnott 1996).
Figure 4.
Figure 4.
Arg85 contacts with the ends of the core-binding site. (A) Sequences and equilibrium binding constants of substrates containing changes at ±7. (B) Interactions between Arg85 and G+7b in the F1 structure. (C) Interaction between Arg85 and A+7b in the F6 structure. The 2Fo − Fc electron density maps in B and C were contoured at 1.5 σ. (D) Comparison of roll angles between +2 and +11 for the F1 and F6 structures.
Figure 5.
Figure 5.
DNA substitutions within the Fis-binding site center. (A) Sequences of Fis-binding substrates together with equilibrium binding constants and half-lives. (B) Minor groove widths of complexes with AT centers. (C) Roll angles over the central segments of complexes with AT centers. (D) Minor groove widths of complexes containing GC substitutions. (E) Roll angles over the central segments of complexes with GC substitutions. (F) DNA parameters over the central 5-bp segments between −2 and +2.
Figure 6.
Figure 6.
DNA substitutions within the ±(3–4) positions of the Fis-binding site. (A) Sequences of Fis-binding substrates and equilibrium binding constants. (B) View of the Asn84–DNA contacts in the right half of the F1 complex. (C) The Asn84–DNA contacts over the same region in the F18 complex (G-to-C mutation at +4). (D) The Asn84–DNA contacts over the same region in the F21 complex (T-to-G mutation at +3). (E) Plot of roll angles over the right half of the F1, F18, F21, and F23 DNA structures. (F) Minor groove widths for the F1, F18, and F23 structures. (G) Sequence and equilibrium binding constants for Fis-wt and Fis-N84A binding to F19 containing the symmetrical mutations at ±4 that position a T proximal to the N84 side chain. (H) Structure of the Fis–F23 complex highlighting Asn84 over the region containing a G-to-T mutation at +4, as well as a T-to-G change at +3. The van der Waals surface of the T+4 methyl, which disrupts the position of the Asn84 side chain, is displayed. (I) EMSAs on the F19 duplex oligonucleotide using twofold increasing amounts of Fis-wt and N84A mutant proteins beginning with 0.08 nM. On the right are binding isotherms of Fis-wt (circles) and Fis-N84A (squares) binding to the F1 (orange) or F19 (blue) DNA substrates.
Figure 7.
Figure 7.
Structures of in silico DNA models. (A) Summary of DNA features of in silico models representing the F1 complex. RMSD (angstroms) refers to a least-squares alignment of the models with DNA residues −12 to +12 of the F1 complex crystal structure. The minimum minor groove width is the narrowest distance within the DNA center. F1 crystal is the DNA from the crystal structure, B DNA is the F1 sequence DNA model constructed from average B DNA parameters, and F1 model is the DNA model constructed from all 12 parameters from the F1 crystal structure (see the Materials and Methods); the remaining entries reflect DNA constructed using average B DNA parameters, except for the listed parameter(s) from the F1 crystal structure. Thus, “Roll only” incorporates the individual rolls from the crystal structure, and “Roll, Twist, and Slide” incorporate those three values from each base-pair step in the crystal structure. (B) Plot of minor groove widths of the F1 crystal structure (orange), the F1 model using all 12 parameters (magenta), and the model using only roll, twist, and slide (green). Additional plots of models listed in A are provided in Supplemental Figure S7. (C) Minor groove widths of models representing the intrinsic structure of the F1 sequence using sequence-specific values for roll, twist, slide, shift, tilt, and rise (orange), or values for only roll, twist, and slide (green), together with average values for the other parameters. (D) Minor groove widths of models representing the intrinsic structure of the Fis-binding sites with different AT centers. Analogous plots of the DNA in the Fis-bound complex are in Figure 5B. (E) Minor groove widths of models representing the intrinsic structure of the Fis-binding sites with central G/C substitutions. Plots of the F27 and F29 DNA in Fis-bound complexes are in Figure 5D. In D and E DNA models were constructed using the six sequence-specific dinucleotide parameters. (F, left) Docking of the Fis dimer onto canonical B DNA such that the HTH on the right side is inserted into the major groove, but the left HTH clashes with the DNA. (Middle) Docking of Fis onto DNA constructed with the twist and slide parameters of the F1 complex, achieving a partial fit of both HTH motifs into the major groove. (Right) Addition of the F1 roll parameters to the DNA in the middle panel, generating a close fit (RMSD = 0.8 Å) with the DNA in the F1 crystal structure.
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