The HU–DNA binding interaction probed with UV resonance Raman spectroscopy: Structural elements of specificity

HU, a multifunctional DNA-binding protein, participates in several cellular functions including growth, recombination, replication and transposition (Drlica and Rouviere-Yaniv 1987). Originally identified as a histone-like protein (Rouviere-Yaniv and Gros 1975), more recent studies implicate HU as a DNA chaperone (Travers 1993) or architectural protein. HU binding mediates DNA looping or bending, which facilitates the formation of multiprotein complexes (Lavoie and Chaconas 1993; Lavoie et al. 1996; Aki and Adhya 1997; Lia et al. 2003). Although HU protein participates in specific functions within the cell and binds to specific DNA sites, as in the case of Gal repression and Mu transposition, it does not exhibit sequence specific recognition of DNA. HU, a heterodimer in Escherichia coli, binds with high affinity and 1:1 stoichiometry (HU dimer:DNA duplex) to discontinuous or bent DNA conformations, such as gapped or nicked DNA; whereas the binding of HU to linear duplex DNA is characterized by micromolar affinities and protein:DNA ratios greater than 1:1 (Castaing et al. 1995; Lavoie et al. 1996; Kamashev et al. 1999; Kobryn et al. 1999; Pinson et al. 1999; Kamashev and Rouviere-Yaniv 2000; Wojtuszewski et al. 2001). The structures of the 1:1 HU–DNA complexes have been successfully modeled using the IHF–DNA cocrystal structure, in which the protein induces a bend of 160° (Lavoie et al. 1996; Rice et al. 1996; Kamashev et al. 1999). The modeling of these complexes indicates that in certain instances HU can bind DNA duplexes site-specifically and induce a bend in a manner similar to IHF. The recent Anabena HU–DNA X-ray crystal structure (Swinger et al. 2003) reveals that the protein-induced bend is variable and not necessarily as large as in the IHF–DNA structure. IHF and HU interact with DNA through some common motifs such as the intercalation of Pro residues to induce bending or kinking of the DNA and interaction of the C terminus with the DNA (Rice et al. 1996; Swinger et al. 2003). In both structures protein-induced bending leads to the wrapping of the DNA around the protein α-helical body. The nanomolar binding affinities observed with flexible or discontinuous DNA substrates and the micromolar binding affinities observed with linear DNA suggests that HU dimer exhibits different modes of DNA binding, which are governed by structure and not sequence, as has been recently described for HMG1 (Murphy and Churchill 2000). Further evidence for an alternate mode of HU binding to DNA has been observed by scanning force microscopy, in which HU binding leads to DNA elongation rather than compaction (Dame and Goosen 2002). The molecular basis that underlies these differences in HU binding interactions is not known. The goal of this study is to identify structural elements important in binding and recognition by examining three different HU–DNA complexes using UV resonance Raman spectroscopy (UVRR): two higher affinity complexes that exhibit protein-induced bending are compared with a low-affinity complex that exhibits no bending. The three complexes have been previously characterized by fluorescence spectroscopy and analytical ultracentrifugation (Wojtuszewski et al. 2001; Wojtuszewski and Mukerji 2003), and were chosen for further study using UVRR spectroscopy because of the observed differences in binding affinity and the amount of protein-induced bending. In the higher affinity complexes (Ka ~5 μM−1) the duplexes are 20 bp long and contain an A4T4 repeating sequence either in the 5′ to 3′ direction or in the 3′ to 5′ direction, hereafter referred to as A4–20 and T4–20, respectively. The orientation of the A-tract leads to a marked difference in structural and energetic properties, which were attributed to DNA bending (Hagerman 1986; Burkhoff and Tullius 1988; Haran and Crothers 1989). Fluorescence resonance energy transfer measurements (FRET) determined a bend angle of 45° and 12° for the A4–20 and T4–20 duplexes and protein binding increased the total bend to 70° and 62°, respectively (Wojtuszewski and Mukerji 2003). Also examined was HU binding to a 13-bp duplex, the sequence of which is based on the IHF H1 consensus binding sequence. HU binds to this sequence with low affinity (0.5 μM−1), a 2:1 stoichiometry and little to no induced bending, as determined by fluorescence intensity measurements (Wojtuszewski et al. 2001). UVRR spectroscopy is now used to probe the structural elements of these complexes to identify their similarities and differences particularly with respect to binding affinity. This study elucidates molecular details of the HU–DNA interaction, which could not be discerned from the previous fluorescence experiments and provides a framework from which the structural recognition of DNA by HU can be understood. Previously, UV resonance Raman spectroscopy has been used successfully to analyze proteins (Harada and Takeuchi 1986; Austin et al. 1993) and DNA (Fodor and Spiro 1986; Toyama et al. 1991, 1993; Mukerji et al. 1995, 1996, 1998; Sokolov et al. 2000; Mukerji and Williams 2002), but the technique has only been sparingly applied to the analysis of protein–DNA interactions. In contrast, Raman spectroscopy has been successfully applied to the study of DNA structure and protein–DNA interactions, and many previously identified conformational markers are used to analyze the current data (Thomas and Tsuboi 1993; Peticolas 1995; Thomas 1999). However, conducting the experiment in a resonance enhanced manner affords several advantages including the differential investigation of the protein and DNA moieties in the complex and the ability to use concentrations that are only modestly increased relative to those used to characterize the binding interaction by other methods (Wojtuszewski et al. 2001; Wojtuszewski and Mukerji 2003). In this study, excitation wavelengths of 210 and 215 nm are used to probe HU secondary structure through the protein amide backbone modes, and the local environment of Phe and Pro residues, respectively (Fig. 1 ▶). An excitation wavelength of 215 nm also enhances deoxyribose phosphate backbone modes, DNA carbonyl stretching vibrations and ring-stretching vibrations, the intensities of which are a reliable measure of base stacking. Structural details of the complexes can be inferred from changes in frequency or intensity of these modes upon complexation. Figure 1. Anabena HU bound to DNA. HU monomers are shown in gray and black to indicate the heterodimeric nature of E. coli HU. The four Pro residues present in E. coli HU, located at positions 63 and 77 for each monomer, are shown in maroon. The six Phe residues ... The current study reveals that the bent complexes are very similar in structure, despite the fact that the A4–20 duplex is prebent. Significant structural features of these complexes, elucidated by UVRR, include increased protein secondary structure content, Pro intercalation to induce bending and a distorted DNA helix with an opened minor groove. In contrast, these structural features are reduced or nonexistent in the linear, low-affinity HU2:H1–13 complex. These features are consistent with a model of a mutually induced fit as originally proposed by Rice and coworkers (Swinger et al. 2003).