Structure-based analysis of HU-DNA binding

Abstract

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

Figure 1. IHF-and AHU-DNA cocrystal structures

(a) Stereoview of a superposition of the IHF- and AHU-DNA complexes. IHF protein is shown in grey and white while the IHF DNA is pink (1IHF). AHU is gold and the bound DNA is green (1P71). Prolines at the tips of arm-like β -ribbon extension are in yellow. (b) AHU-DNA complex. The AHU-DNA complex (1P71) is color coded as in Table 1. The protein subunits are gold and orange and the intercalating prolines are in yellow. Canonical DNA is blue while unpaired bases are green (stacked) or grey (flipped) and mismatches are pink. Part b reprinted from Figure 3 of Swinger and Rice, 2003.

Figure 2. Origin of DNA duplexes used in this binding study

(a) IHF’s cognate site, H’. The IHF consensus site is underlined in green. The black arrows show the location of backbone nicks placed 9 base pairs apart in an effort to phase AHU binding for homogeneous crystal formation. The dashed arrow shows the location of the nick in the IHF-DNA crystal structure (1IHF). (b) DNA observed in an AHU-DNA cocrystal (1P71). Two copies of the top right oligonucleotide bound to each other forming a duplex with 4 unpaired Ts and 3 T • T mismatches. Color coding is as follows: green, analogous base in crystal structure is stacked; grey, analogous base in crystal structure is flipped; pink, T • T base pair (c) DNA duplex for gel shift experiments. The middle of the site is identical to duplex TR3 from the crystal. The flanking DNA in light blue is identical to DNA from neighboring protein-DNA complexes that contact AHU in the crystal. The black flanking sequence destabilizes a hairpin that forms in its absence and complicates binding experiments.

Figure 3. Representative EMSA experiment

Panel (a) shows a polyacrylamide native gel shift experiment between duplex 6 and AHU. Assays were performed in binding buffer, 20mM Tris–HCl (pH 8.0), 70mM NaCl, 1 μ g/ml of salmon sperm DNA, and 5% glycerol, at 4° C by incubation of the ³²P-labeled DNA various concentrations of protein. Further details are described in Materials and methods. Panel (b) shows the curve fit for data from panel (a) that was used to extract an apparent binding constant. The R value represents the square root of the difference between the total variation (t) and the unexplained variation (u) divided by the total variation or R = √((t-u)/t).

Figure 4. Comparisons of IHF- and AHU- kinked DNA

Panel (a) shows a stereoview of a superposition of kinked DNA from the previously published IHF-DNA structure in pink (1IHF), the sharper of the kinks in the AHU-DNA structure in green (1P78). The tips of the β -ribbon protein arms are shown in white (IHF) and gold (AHU) with the intercalating prolines in yellow. Bases at the kink in these two structures superimpose remarkably well considering the differences in sequences and the presence of an extra T in the AHU structure (marked by an asterisk). The sequences for the portions of the structures shown are in green for AHU and pink for IHF. The asterisk marks the intercalated T. Panel (b) shows a stereoview of a superposition of kinked DNA from a nicked IHF-DNA structure recently deposited to the Protein Data Bank in pink (2HT0) and the AHU-DNA structure in green. The nick in the IHF DNA backbone in this structure is directly at the site of kinking. Proteins are color coded as in panel (a). The superposition of paired bases in these two structures is even closer than that observed in panel (a). The arrow by the IHF sequence in pink marks the location of the nick. This view illustrates that a nick and an extra T similarly relieve strain in the kinked DNA backbone.

Figure 5. EMSA experiment shows that IHF binds to phased single-base insertions

Panel (a) shows a polyacrylamide native gel shift experiment between duplex 4 containing 2 single-T insertions and IHF. Assays were performed in binding buffer, 20mM Tris–HCl (pH 8.0), 70mM NaCl, 1 μ g/ml of salmon sperm DNA, and 5% glycerol, at 4°C by incubation of the ³²P-labeled DNA various concentrations of protein. Further details are described in Materials and methods. Panel (b) is the curve fit used to extract an apparent binding constant of 36nM for the gel in panel (a). After three repetitions of the experiment, an apparent K_d of 42±6nM was determined as reflected in Table 1.