Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.     

Molecular dynamics of amphotericin B. II. Dimer in water

Molecular dynamics simulations were performed for a dimer of the antifungal antibiotic, amphotericin B, in water. In the first step of the work three appropriately selected versions of the dimer structure were taken into consideration. In each version antibiotic molecules were placed antiparallel with polar and ionizable groups outside the hydrophobic core formed by polyene chromophores. During short dynamic simulations versions of the dimer structure were compared in respect of the energy of dimerization. The highest energy was observed for the structure in which polyene chromophores superimposed each other as much as possible and this version was subjected to the main simulation. The analysis of 66 snapshot geometries stored during 33 ps dynamic trajectory allowed us to draw three main conclusions: (i) the relative orientation of the amino-sugar moiety and chromophore as well as conformation of the antibiotic macrolide ring were different in both molecules and could exhibit dynamic changes, (ii) the dimer structure exhibited intrinsic asymmetry which could be responsible for characteristic circular dichroism spectra of the aggregated form of the antibiotic, (iii) relatively high stability of the dimer structure resulted not only from hydrophobic interactions between chromophores but also from hydrogen bonds networks that were observed around polar terminals of antibiotic molecules. Implications of these features of the dimer structure for its susceptibility on the ionic state of carboxyl and/or amino groups are also discussed.     

Interaction of human SRY protein with DNA: a molecular dynamics study

Molecular dynamics simulations have been conducted to study the interaction of human sex-determining region Y (hSRY) protein with DNA. For this purpose, simulations of the hSRY high mobility group (HMG) domain (hSRY-HMG) with and without its DNA target site, a DNA octamer, and the DNA octamer alone have been carried out, employing the NMR solution structure of hSRY-HMG-DNA complex as a starting model. Analyses of the simulation results demonstrated that the interaction between hSRY and DNA was hydrophobic, just a few hydrogen bonds and only one water molecule as hydrogen-bonding bridge were observed at the protein-DNA interface. These two hydrophobic cores in the hSRY-HMG domain were the physical basis of hSRY-HMG-DNA specific interaction. They not only maintained the stability of the complex, but also primarily caused the DNA deformation. The salt bridges formed between the positive-charged residues of hSRY and phosphate groups of DNA made the phosphate electroneutral, which was advantageous for the deformation of DNA and the formation of a stable complex. We predicted the structure of hSRY-HMG domain in the free state and found that both hSRY and DNA changed their conformations to achieve greater complementarity of geometries and properties during the binding process; that is, the protein increased the angle between its long and short arms to accommodate the DNA, and the DNA became bent severely to adapt to the protein, although the conformational change of DNA was more severe than that of the hSRY-HMG domain. The sequence specificity and the role of residue Met9 are also discussed.     

The ligand insertion hypothesis in the genomic action of steroid hormones

Gene regulation by steroids is tightly coupled to hormone concentration and stereochemistry. A key step is binding of hormones to receptors which interact with consensus DNA sequences known as hormone response elements (HREs). The specificity and strength of hormone binding do not correlate well with hormonal activity suggesting an additional step involving recognition of ligand by the gene. Stereospecific fit of hormones between base pairs and correlation of fit with hormonal activity led to the proposal that such recognition involves insertion of hormone into DNA. Here, the feasibility of insertion was investigated using computer models of the glucocorticoid receptor DNA binding domain bound to its HRE. The site reported to accommodate glucocorticoids was found in the HRE and was exposed to permit unwinding at this locus. The resulting cavity in the unwound DNA/receptor interface fit cortisol remarkably well; cortisol formed hydrogen bonds to both the receptor and DNA. Current experimental evidence is generally consistent with ligand binding domains of receptors undergoing a conformational change which facilitates transfer of the ligand into the unwound DNA/receptor interface. We propose this step is rate limiting and alterations in receptor, DNA or hormone which attenuate insertion impair hormonal regulation of gene function.     

A model of water structure inside the HLA-A2 peptide binding groove

Based on molecular dynamics simulations, it is proposed that water within the binding groove of the human MHC class I molecule HLA-A2 plays a role in the formation of its complex with the influenza matrix protein (residues 58-66; GILGFVFTL) peptide. In these simulations, a loosely structured network of water molecules is present in the binding groove between the peptide and the MHC molecule, and may be important in completing the peptide-MHC interface. In two independent 400 ps simulations where groove-based water molecules were included, the peptide remained essentially in the conformation observed in the crystal structure. In contrast, in a 400 ps simulation in which no water molecules were placed between the peptide and the MHC molecule, the crystal structure conformation was rapidly lost. The basis for this behavior appears to be that the groove-based water molecules help to maintain the appropriate orientation of the Arg-97 side chain of HLA-A2 and, in turn, the conformation of the central part of the peptide.     

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.     

Interstitial laser photocoagulation of normal lung parenchyma in rats

Homeodomains are one of the key families of eukaryotic DNA-binding motifs and provide an important model system for studying protein-DNA interactions. We have crystallized the Antennapedia homeodomain-DNA complex and solved this structure at 2.4 A resolution. NMR and molecular dynamics studies had implied that this homeodomain achieves specificity through an ensemble of rapidly fluctuating DNA contacts. The crystal structure is in agreement with the underlying NMR data, but our structure reveals a well-defined set of contacts and also reveals the locations and roles of water molecules at the protein-DNA interface. The synthesis of X-ray and NMR studies provides a unified, general model for homeodomain-DNA interactions.     

Solution structure of nodularin. An inhibitor of serine/threonine-specific protein phosphatases

The three-dimensional solution structure of nodularin was studied by NMR and molecular dynamics simulations. The conformation in water was determined from the distance and dihedral data by distance geometry and refined by iterative relaxation matrix analysis. The cyclic backbone adopts a well defined conformation but the remote parts of the side chains of arginine as well as the amino acid derivative Adda have a large spatial dispersion. For the unusual amino acids the partial charges were calculated and nodularin was subjected to molecular dynamic simulations in water. A good agreement was found between experimental and computational data with hydrogen bonds, solvent accessibility, molecular motion, and conformational exchange. The three-dimensional structure resembles very closely that of microcystin-LR in the chemically equivalent segment. Therefore, it is expected that the binding of both microcystins and nodularins to serine/threonine-specific protein phosphatases is similar on an atomic level.     

DNA binding of hypothalamic nuclear proteins on estrogen response element and preproenkephalin promoter: modification by estrogen

Preproenkephalin (PPE) gene expression is specifically induced by estrogen in hypothalamus of ovariectomized (OVX) females, better than in male rats. To study estrogen actions on gene regulation, we have presently characterized protein-DNA interactions by use of a consensus estrogen response element (ERE) and a putative ERE from PPE gene, with nuclear extracts from hypothalamus. By use of the electrophoretic mobility shift assay (EMSA), ERE binding activity was detected in nuclear extracts from neuronal tissues including hypothalamus, hippocampus, striatum, cerebellum and frontal cortex, and non-neuronal tissues such as pituitary and uterus, but not lung of OVX female rats with a consensus ERE, as well as a 129-bp PCR fragment from PPE promoter and a hairpin oligonucleotide that contains a putative ERE of the rat PPE gene. The ERE binding was eliminated by the addition of specific ERE-containing oligonucleotide, but not control oligonucleotides. Protein and DNA associated and dissociated very rapidly. By use of supershift assay, interactions of estrogen receptor with ERE were demonstrated in hypothalamic nuclear extracts. The initial levels of specific ERE binding in the hypothalamic nuclear extracts were comparable between castrated male and OVX female rats. However, estrogen treatment, either estradiol or estradiol benzoate, produced a rapid and tissue-specific induction of a slow mobility complex of ERE binding in hypothalamic nuclear extracts from females, better than in male rats, presumably from other associated factors, or a conformational change or other posttranslational modifications. This estrogen-induced slow mobility complex of ERE binding in hypothalamus was not observed after treatment with progesterone or tamoxifen. These results suggest that specific ERE binding is present in rat hypothalamic nuclear proteins, which may contribute to the upregulation of PPE gene expression by estrogen, and that the sexually differentiated action of estrogen may be related to an estrogen-induced conformational change, but not to the initial level of ERE-binding activity.     

High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA

The crystal structure of the paired homeodomain bound to DNA as a cooperative dimer has been determined to 2.0 A resolution. Direct contacts between each homeodomain and the DNA are similar to those described previously. In addition, an extensive network of water molecules mediates contacts between the recognition helix and the DNA major groove. Several symmetrical contacts between the two homeodomains underlie the cooperative interaction, and deformations in the DNA structure are necessary for the establishment of these contacts. Comparison with structures of homeodomains bound monomerically to DNA suggests that the binding of a single paired homeodomain can introduce these DNA distortions, thus preparing a template for the cooperative interaction with a second homeodomain. This study shows how the paired (Pax) class homeodomains have achieved cooperativity in DNA binding without the assistance of other domains, thereby enabling the recognition of target sequences that are long enough to ensure specificity.     

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.     

Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.     

Structural and dynamic effects of point mutations in the recognition helix of the glucocorticoid receptor DNA-binding domain

We have studied the wild type and two variants of the glucocorticoid receptor DNA-binding domain (GRDBD): in one variant the three residues (the 'P-box' in GRDBD) that are essential for the discrimination between GREH and EREH are mutated to those in the estrogen receptor DBD (GRDBDega) and the other variant is a point mutation of one P-box residue, Ser459Gly (GRDBDggv). Molecular dynamics simulations (0.5-0.7 ns) have been performed on the GRDBDs, free in solution as well as in complex with the half-site response elements of the glucocorticoid (GREH) and estrogen (EREH) receptors. The residues which are central when forming the protein dimer interface in GRE-(GRDBD)2 (the 'D-box') were found to have different conformations in the different GRDBD-DNA complexes. This is consistent with experimental results showing that the cooperativity of dimeric GRDBD binding to DNA strongly depends on both the response element and the P-box residues. In our simulations the structures of GREH-GRDBDgsv (i.e. wild-type) and GREH-GRDBDggv were more similar to each other than to the respective GRDBDs bound to EREH. This is due to a thymine methyl group which is present in the major groove of the GREH and prevents the first zinc coordinating subdomain in GRDBD to approach GREH, but which is absent in EREH. Thus, EREH-GRDBD is able to respond more to the Ser459Gly mutation than GREH-GRDBD.     

Binding of Net-Fla, a netropsin-flavin hybrid molecule, to DNA: molecular mechanics and dynamics studies in vacuo and in water solution

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A. T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the drug-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.     

RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations

Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.     

Affinity selective isolation of ligands from peptide libraries through display on a lac repressor "headpiece dimer"

DNA binding by the Escherichia coli lac repressor is mediated by the approximately 60 amino acid residue 'headpiece' domain. The dimer of headpiece domains that binds to the lac operator is normally formed by association of the much larger approximately 300 amino acid residue C-terminal domain. We have used in vitro selection to isolate 'headpiece dimer' molecules containing two headpiece domains connected via a short peptide linker. These proteins bind plasmid molecules with sufficient stability to allow association of a peptide epitope displayed at the C terminus of the headpiece dimer with the plasmid encoding that peptide. Libraries of peptides displayed on the C terminus of a headpiece dimer can be screened for specific receptor ligands by affinity enrichment of peptide-headpiece dimer-plasmid complexes using an immobilized receptor. After each round of enrichment, transformation of E. coli with recovered plasmids permits amplification of the selected population. After several rounds of enrichment, sequencing of individual clones reveals the structure of the selected peptides. Headpiece dimer libraries allow selection of peptide ligands of higher average affinity than similar libraries based on the intact lac repressor. Interestingly, the presence of the lac operator is not required for plasmid binding by the headpiece dimer protein.     

Homology model for the ligand-binding domain of the human estrogen receptor

We have modeled the ligand-binding domain (LBD) of the human estrogen receptor protein (hER) by homology to the known crystal structure of the LBD of the alpha isoform of human retinoate-X receptor (hRX). Alignment of hER with members of the nuclear receptor superfamily defined probable secondary structures which we used to constrain backbone torsion angles and hydrogen bonds. From published studies we identified key interactions between hER and estradiol to use to dock the hormone in its ligand-binding pocket. Since the hRX crystal structure corresponds to the unliganded form of the LBD, we adopted the "mousetrap" mechanism proposed by Renaud et al to predict the structure of the E2-bound hER. Refinement by molecular dynamics and energy minimization gave a model which matches well the known facts about the estradiol phamacophore. It also provides a possible explanation for how hER discriminates between estradiol and testosterone.     

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.