Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface

Abstract

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236-39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379-401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF-DNA interfaces while the resulting energetic compensation maintains the same net binding energy.

Figure 1.

Crystallographic model of the complex of IHF bound to λ H′ site. The co-ordinate file for this structure, in which a nick in the sequence used to grow crystals (3) has been repaired, was supplied by Phoebe Rice (personal communication). The α and β subunits of IHF are shown in cyan and blue, respectively. Consensus DNA recognition elements are colored: (magenta) A-tract; (orange) ApA steps at proline intercalation sites; (red) direct interaction and (green) remaining base pairs of consensus, WATCAAnnnnTTR motif.

Figure 2.

Electrophoretic mobility-shift assay of IHF binding to oligonucleotide A.2. IHF concentrations in Lanes 1–10 are 0, 20, 40, 60, 81, 99, 120, 165, 201 and 240 nM, respectively. This pseudo-color image was generated by coloring the emission collected through a 520-nm band pass filter green (FAM fluorescence) and coloring the emission collected through a 580-nm band pass filter red (TAMRA fluorescence). With excitation at 488 nm, the unliganded oligonucleotide is green, reflecting only FAM fluorescence. The yellow color of the mobility-shifted band results from a combination of green and red fluorescence, indicating efficient FRET due to the wrapped DNA in the bound complex.

Figure 3.

Panel A shows the pressure FRET ratio baseline data (open circle) and polynomial smoothing curve (solid line) for oligonucleotide A.6 in the absence of IHF compared with unprocessed data for 10 nM DNA and 25 nM IHF (filled square) (10 mM Tris pH 8.0, 100 mM NaCl and 1 mM EDTA). Panel B compares fraction bound for oligonucleotides A.2 (filled diamond) and A.6 (filled square) at 10 nM DNA, 25 nM IHF, i.e. same A.6 data as panel A and same reaction conditions. Solid and dashed curves are the fits and 95% confidence intervals to these individual experiments, using equations (5–7) as described in the text.

Figure 4.

Global analyses of pressure-perturbation curves for oligonucleotides A.2 (solid symbols, broken lines) and A.6 (open symbols and solid lines). The component concentrations are: 3 nM DNA, 9 nM IHF (filled diamond); and 10 nM DNA at 12.5 nM IHF (filled square, open square), at 25 nM IHF (filled circle, open circle) and at 50 nM IHF (filled triangle, open triangle).