Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.     

Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion

The Hin DNA invertase becomes catalytically activated when assembled in an invertasome complex containing two Fis dimers bound to an enhancer segment. The region of Fis responsible for transactivation of Hin contains a mobile beta-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating beta-arm are sufficient to form catalytically competent invertasomes. Analysis of homodimer and heterodimer mixes of different Hin mutants suggests that Fis must activate each subunit of the two Hin dimers that participate in catalysis. These experiments also indicate that all four Hin subunits must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is independent of the catalytic steps of recombination. We propose a molecular model for the invertasome structure that is consistent with current information on protein-DNA structures and the topology of the DNA strands within the recombination complex. In this model, a single Fis activation arm could contact amino acids from both Hin subunits at the dimer interface to induce a conformational change that coordinately positions the active sites close to the scissile phosphodiester bonds.     

On the importance of van der Waals interaction in the groove binding of DNA with ligands: restrained molecular dynamics study

Comparison of interaction energy between an oligonucleotide and a DNA-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics. Distortion in DNA was found for the major groove mode whereas less significant changes for both ligand and DNA were detected for the minor groove binding after molecular dynamics simulation. The conformation of the ligand obtained from the major groove modes resembles that computed with the ligand soaked in water. The van der Waals contact energy was found to be as significant as electrostatic energy and more important for difference in binding energy between these two binding modes. The importance of van der Waals force in groove binding was supported by computations on the complex formed by the repressor peptide fragment from the bacteriophage 434 and its operator oligonucleotide.     

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.     

The solution structure of a fungal AREA protein-DNA complex: an alternative binding mode for the basic carboxyl tail of GATA factors

The solution structure of a complex between the DNA binding domain of a fungal GATA factor and a 13 base-pair oligonucleotide containing its physiologically relevant CGATAG target sequence has been determined by multidimensional nuclear magnetic resonance spectroscopy. The AREA DNA binding domain, from Aspergillus nidulans, possesses a single Cys2-Cys2 zinc finger module and a basic C-terminal tail, which recognize the CGATAG element via an extensive network of hydrophobic interactions with the bases in the major groove and numerous non-specific contacts along the sugar-phosphate backbone. The zinc finger core of the AREA DNA binding domain has the same global fold as that of the C-terminal DNA binding domain of chicken GATA-1. In contrast to the complex with the DNA binding domain of GATA-1 in which the basic C-terminal tail wraps around the DNA and lies in the minor groove, the structure of complex with the AREA DNA binding domain reveals that the C-terminal tail of the fungal domain runs parallel with the sugar phosphate backbone along the edge of the minor groove. This difference is principally attributed to amino acid substitutions at two positions of the AREA DNA binding domain (Val55, Asn62) relative to that of GATA-1 (Gly55, Lys62). The impact of the different C-terminal tail binding modes on the affinity and specificity of GATA factors is discussed.     

Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.     

Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations

Mutations in the p53 tumor suppressor are the most frequently observed genetic alterations in human cancer. The majority of the mutations occur in the core domain which contains the sequence-specific DNA binding activity of the p53 protein (residues 102-292), and they result in loss of DNA binding. The crystal structure of a complex containing the core domain of human p53 and a DNA binding site has been determined at 2.2 angstroms resolution and refined to a crystallographic R factor of 20.5 percent. The core domain structure consists of a beta sandwich that serves as a scaffold for two large loops and a loop-sheet-helix motif. The two loops, which are held together in part by a tetrahedrally coordinated zinc atom, and the loop-sheet-helix motif form the DNA binding surface of p53. Residues from the loop-sheet-helix motif interact in the major groove of the DNA, while an arginine from one of the two large loops interacts in the minor groove. The loops and the loop-sheet-helix motif consist of the conserved regions of the core domain and contain the majority of the p53 mutations identified in tumors. The structure supports the hypothesis that DNA binding is critical for the biological activity of p53, and provides a framework for understanding how mutations inactivate it.     

Primary and tertiary structures of the Fab fragment of a monoclonal anti-E-selectin 7A9 antibody that inhibits neutrophil attachment to endothelial cells

The murine monoclonal IgG1 antibody 7A9 binds specifically to the endothelial leukocyte adhesion molecule-1 (E-selectin), inhibiting the attachment of neutrophils to endothelial cells. The primary and three-dimensional structures of the Fab fragment of 7A9 are reported. The amino acid sequence was determined by automated Edman degradation analysis of proteolytic fragments of both the heavy and light chains of the Fab. The sequences of the two chains are consistent with that of the IgG1 class with an associated kappa light chain with two intrachain disulfide bridges in each of the heavy and light chains. The tertiary structure of the antibody fragment was determined by x-ray crystallographic methods at 2.8 A resolution. The F(ab')2 molecule, treated with dithiothreitol, crystallizes in the space group P2(1) 2(1) 2(1) with unit cell parameters a = 44.5 A, b = 83.8 A, and c = 132.5 A with one Fab molecule in the asymmetric unit. The structure was solved by the molecular replacement method and subsequently refined using simulated annealing followed by conventional least squares optimization of the coordinates. The resulting model has reasonable stereochemistry with an R factor of 0.195. The 7A9 Fab structure has an elbow bend of 162 degrees and is remarkably similar to that of the monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody Fab fragment. The 7A9 antigen combining site presents a groove resembling the structure of the anti-ICAM-1 antibody, and other antibodies raised against surface receptors and peptides. Residues from the six complementary determining regions (CDRs) and framework residues form the floor and walls of the groove that is approximately 22 A wide and 8 A deep and that is lined with many aromatic residues. The groove is large enough to accommodate the loop between beta-strands beta4 and beta5 of the lectin domain of E-selectin that has been implicated in neutrophil adhesion (1).     

NMR spectroscopic studies of the DNA-binding domain of the monomer-binding nuclear orphan receptor, human estrogen related receptor-2. The carboxyl-terminal extension to the zinc-finger region is unstructured in the free form of the protein

Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 (hERR2) belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers. A carboxyl-terminal extension (CTE) to the zinc-finger domain has been implicated to be responsible for determining the stoichiometry of binding by a nuclear receptor to its response element. To better understand the mechanism by which DNA specificity is achieved, the solution structure of the DNA-binding domain of hERR2 (residues 96-194) consisting of the two putative zinc fingers and the requisite 26-amino acid CTE was analyzed by multidimensional heteronuclear magnetic resonance spectroscopy. The highly conserved zinc-finger region (residues 103-168) has a fold similar to those reported for steroid and retinoid receptors, with two helices that originate from the carboxyl-terminal ends of the two zinc fingers and that pack together orthogonally, forming a hydrophobic core. The CTE element of hERR2 is unstructured and highly flexible, exhibiting nearly random coil chemical shifts, extreme sensitivity of the backbone amide protons to solvent presaturation, and reduced heteronuclear (1H-15N) nuclear Overhauser effect values. This is in contrast to the dimer-binding retinoid X and thyroid hormone receptors, where, in each case, a helix has been observed within the CTE. The implications of this property of the hERR2 CTE are discussed.