Structural perturbations induced in linear and circular DNA by the architectural protein HU from Bacillus stearothermophilus

HU is a small DNA-binding protein of eubacteria that is believed to induce or stabilize bending of the double helix and mediate nucleoid compaction in vivo. Although HU does not bind preferentially to specific DNA sequences, it is known to have high affinity for DNA sites containing structural anomalies, such as unpaired or mismatched bases, nicks, and four-way junctions. We have employed Raman spectroscopy to further investigate the structural basis of HU-DNA recognition in solution. Experiments were carried out on the homodimeric HU protein of Bacillus stearothermophilus (HUBst) and a 222-bp DNA fragment, which was isolated in linear (DNA(L222)) and circular (DNA(C222)) forms. In the absence of bound HUBst the Raman signatures of DNA(L222) and DNA(C222) are nearly superimposable, indicating that circularization produces no substantial change in the local B-DNA conformation. Conversely, the Raman signatures of DNA(L222) and DNA(C222) are perturbed significantly and specifically by HUBst binding. The HUBst-induced perturbations are markedly greater for the circularized DNA target. These results support an opportunistic molecular mechanism, in which HU binding is facilitated by intrinsic nonlinearity or flexibility in the DNA target. We propose that DNA segments which are bent or predisposed toward bending provide the high-affinity sites for HU attachment and nucleoid condensation. This model is consistent with the wide range of DNA bending angles reported in crystal structures of HU-DNA complexes.     

Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.

The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI.     

Binding of the estrogen receptor to DNA. The role of waters.

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.     

The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition

AbrB is a Bacillus subtilis protein responsible for regulating a diverse array of unrelated genes during periods of sub-optimal growth conditions. DNA binding by AbrB is unique in that sequence recognition is specific, yet no obvious consensus sequence of bound promoter regions is apparent. The N-terminal domain is a recently characterized representative of a novel class of DNA-binding proteins that possess a looped-hinge helix DNA-binding topology. Although the structural characterization of this DNA-binding topology contributed to an understanding of the architectural basis for recognition of DNA target sequences, specific mechanisms responsible for promiscuity in DNA sequence recognition still were not apparent. Analysis of (15)N backbone relaxation parameters shows that dynamic motion of regions directly linked to DNA binding show concerted motion on the microsecond-millisecond timescale. Furthermore, dynamic motion of the hinge region suggests that the DNA-binding region is capable of conformational orientations that allow it to accommodate DNA sequence variability in the cognate binding sites.     

Recognizing DNA

It has become clear that there is no simple 'code' for protein-DNA recognition and that selecting an optimal binding sequence along the DNA double helix corresponds to more than simply forming a set of specific hydrogen bonds or steric interactions. However, it has been difficult to characterize the so-called indirect components of recognition. While DNA deformation certainly underlies indirect recognition, it is not easy to determine how local fine structure and deformability depend on base sequence or exactly what percentage of recognition should be attributed to such factors. Molecular modelling can help to develop these ideas into a quantitative model, provided the calculations can be carried out fast enough to enable a comprehensive survey of base-sequence effects. I present here some recent results from our group and their consequences for improving our understanding of protein-DNA binding, and their potential for predicting, and eventually modulating, protein-DNA binding.     

An analysis of the relationship between hydration and protein-DNA interactions.

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR.

Integration Host Factor, IHF, is an E. coli DNA binding protein that imposes a substantial bend on DNA. Previous footprinting studies and bending assays have characterized several recognition sequences in the bacterial and lambda phage genome as unique in the way they are bound by IHF. We have chosen one of the lambda phage sites, H1, for study because it presents a small yet sequence-specific substrate for NMR analysis of the complex. A 19 base-pair duplex, H19, corresponding to the recognition sequence at the H1 site was constructed by isotopically labeling one of the strands with 15N. (1H, 15N) heteronuclear NMR experiments aided in assigning the imino proton resonances of the DNA alone and in complex with IHF. The NMR results are consistent with a mode of binding observed in the recent crystal structure of IHF bound to another of its sites from the lambda phage genome. Additionally, the dramatic change that IHF imposes on the imino proton chemical shifts is indicative of a severe deviation from canonical B-DNA structure. In order to understand the dynamic properties of the DNA in the complex with IHF, the exchange rates of the imino protons with the solvent have been measured for H19 with and without IHF bound. A drastic reduction in exchange is observed for the imino protons in the IHF bound DNA. In the DNA-protein complex, groups of adjacent base-pair exchange at the same rate, and appear to close more slowly than the rate of imino proton exchange with bulk water, since their exchange rate is independent of catalyst concentration. We infer that segments of the double helix as large as 6 bp open in a cooperative process, and remain open much longer than is typical for opening fluctuations in naked duplex DNA. We discuss these results in terms of the specific protein-DNA contacts observed in the crystal structure.     

GATA-1 bends DNA in a site-independent fashion

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.     

Rearrangement of side-chains in a Zif268 mutant highlights the complexities of zinc finger-DNA recognition.

Structural and biochemical studies of Cys(2)His(2) zinc finger proteins initially led several groups to propose a "recognition code" involving a simple set of rules relating key amino acid residues in the zinc finger protein to bases in its DNA site. One recent study from our group, involving geometric analysis of protein-DNA interactions, has discussed limitations of this idea and has shown how the spatial relationship between the polypeptide backbone and the DNA helps to determine what contacts are possible at any given position in a protein-DNA complex. Here we report a study of a zinc finger variant that highlights yet another source of complexity inherent in protein-DNA recognition. In particular, we find that mutations can cause key side-chains to rearrange at the protein-DNA interface without fundamental changes in the spatial relationship between the polypeptide backbone and the DNA. This is clear from a simple analysis of the binding site preferences and co-crystal structures for the Asp20-->Ala point mutant of Zif268. This point mutation in finger one changes the specificity of the protein from GCG TGG GCG to GCG TGG GC(G/T), and we have solved crystal structures of the D20A mutant bound to both types of sites. The structure of the D20A mutant bound to the GCG site reveals that contacts from key residues in the recognition helix are coupled in complex ways. The structure of the complex with the GCT site also shows an important new water molecule at the protein-DNA interface. These side-chain/side-chain interactions, and resultant changes in hydration at the interface, affect binding specificity in ways that cannot be predicted either from a simple recognition code or from analysis of spatial relationships at the protein-DNA interface. Accurate computer modeling of protein-DNA interfaces remains a challenging problem and will require systematic strategies for modeling side-chain rearrangements and change in hydration.     

The role of residue 50 and hydration water molecules in homeodomain DNA recognition

We conducted molecular dynamics simulations on several wild-type and mutant homeodomain-DNA complexes to investigate the role of residue 50 in homeodomain-DNA interaction and the behavior of interfacial hydration water. Our results suggest that this residue interacts more favorably with its consensus sequence and thus plays a considerable role in DNA recognition. However, residue 50 was not responsible for DNA recognition alone. Other residues in the vicinity could interact with residue 50 in cooperation upon DNA binding. We also found the lifetime for some water in the protein-DNA interface can be as high as nanoseconds and that a few well-conserved sites for water-mediated hydrogen bonds from protein to DNA are occupied by high-mobility hydrating waters.     

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.     

How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures

Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2. They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units. Despite these similarities, TRF1 and TRF2 have different functions at telomeres. The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 angstroms resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p. Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions. The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.     

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.