Molecular beacon-equilibrium cyclization detection of DNA-protein complexes

Molecular beacon detection of equilibrium cyclization (MBEC) is a novel, high sensitivity technique that can allow DNA-protein complex formation to be studied under diverse conditions in a cost effective and rapid manner that can be adapted to high throughput screening. To demonstrate the ease and utility of applying MBEC to the investigation of the K(D) values of protein-DNA complexes, the sequence-specific Escherichia coli integration host factor (IHF) protein has been used as a test system. Competition between a labeled MBEC DNA construct and unlabeled duplex DNA for IHF binding allows the determination of K(D) values as a function of the DNA duplex sequence. This allows sequence specificity to be monitored while using only a single molecular beacon-labeled DNA. The robustness of MBEC for monitoring protein-DNA complex formation has been further demonstrated by determining the K(D) values as a function of salt concentration to investigate the net number of salt bridges formed in sequence-specific and -nonspecific IHF-DNA complexes. These MBEC results have been compared with those from other approaches.     

Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.     

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding.

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA. At low to moderate [K(+)] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size. In this [K(+)] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K(+)]. Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K(+)]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K(+)]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K(+)] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.     

Insights into the Structure,Correlated Motions,and Electrostatic Properties of Two HIV-1 gp120 V3 Loops

The V3 loop of the glycoprotein 120 (gp120) is a contact point for cell entry of HIV-1 leading to infection. Despite sequence variability and lack of specific structure, the highly flexible V3 loop possesses a well-defined role in recognizing and selecting cell-bound coreceptors CCR5 and CXCR4 through a mechanism of charge complementarity. We have performed two independent molecular dynamics (MD) simulations to gain insights into the dynamic character of two V3 loops with slightly different sequences, but significantly different starting crystallographic structures. We have identified highly populated trajectory-specific salt bridges between oppositely charged stem residues Arg9 and Glu25 or Asp29. The two trajectories share nearly identical correlated motions within the simulations, despite their different overall structures. High occupancy salt bridges play a key role in the major cross-correlated motions in both trajectories, and may be responsible for transient structural stability in preparation for coreceptor binding. In addition, the two V3 loops visit conformations with similarities in spatial distributions of electrostatic potentials, despite their inherent flexibility, which may play a role in coreceptor recognition. It is plausible that cooperativity between overall electrostatic potential, charged residue interactions, and correlated motions could be associated with a coreceptor selection and binding.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.     

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

Homeodomains are a class of helix-turn-helix DNA-binding protein motifs that play an important role in the control of cellular development in eukaryotes. They fold in a three alpha-helix structural module, where the third helix is the recognition helix that fits into the major groove of DNA. Structural analysis of the members of the homeodomain family led to the identification of interactions likely to stabilize the protein domains. Linking the helices pairwise, three salt bridges were found to be well preserved within the family. Also well conserved were two cation-pi interactions between aromatic and positively charged side chains. To analyze the structural role of the salt bridges, molecular dynamics simulations (MD) were carried out on the wild-type homeodomain from the Drosophila paired protein (1fjl) and on three mutants, which lack one or two salt bridges and mimic natural mutations in other homeodomains. Analysis of the trajectories revealed only small structural rearrangements of the three helices in all MD simulations, thereby suggesting that the salt bridges have no essential stabilizing role at room temperature, but rather might be important for improving thermostability. The latter hypothesis is supported by a good correlation between the melting midpoint temperatures of several homeodomains and the number of salt bridges and cation-pi interactions that connect secondary structures.     

The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers.

Integration host factor (IHF) is a heterodimeric protein from Escherichia coli which specifically binds to an asymmetric consensus sequence. We have isolated the individual subunits of IHF, HimA and HimD, and show that an active IHF protein can be reconstituted from these subunits. The HimA and HimD polypeptides alone are capable of specifically recognizing the same ihf sequence. The mobilities of the protein-DNA complexes in a gel-retardation assay suggest that the proteins bind as homodimers. The stability of the HimD-DNA complex is approximately 100-fold lower than that of the IHF-DNA complex. The HimA-DNA complex is even less stable and is only observed when a large excess of HimA is used. This instability is possibly due to the inability of HimA to form stable homodimers. By domain swapping between HimA and HimD, we have constructed an IHF fusion protein which has the putative DNA-binding domains of only HimA. This fusion protein forms stable dimers and makes specific protein-DNA complexes with a high efficiency. A comparable fusion protein with only the DNA-binding domains of HimD forms less stable complexes, suggesting that sequence-specific contacts between IHF and the ihf consensus are mainly provided by the HimA subunit.     

The temperature dependence of salt-protein association is sequence specific

Molecular dynamics (MD) simulations are used to probe the origin of the unexpected temperature dependence of salt accumulation in the C-terminal region of the protein human lymphotactin. As in previous MD simulations, sodium ions accumulate in an enhanced manner near the C-terminal helix at the lower temperature, while the temperature dependence of chloride accumulation is much weaker and slightly positive. In a designed mutant in which all positively charged residues in the C-terminal helix are replaced with neutral polar groups (Ser), the unexpected temperature dependence of the sodium ions is no longer observed. Therefore, these simulations convincingly verified the previous hypothesis that the temperature dependence of ion-protein association is sensitive to the local sequence. This is explained qualitatively in terms of the entropy of association between charged species in solution. These findings have general implications for the interpretation of thermodynamic quantities associated with binding events where ion release is important, such as protein-DNA interactions.     

Peptide Binding to a PDZ Domain by Electrostatic Steering via Nonnative Salt Bridges

We have captured the binding of a peptide to a PDZ domain by unbiased molecular dynamics simulations. Analysis of the trajectories reveals on-pathway encounter complex formation, which is driven by electrostatic interactions between negatively charged carboxylate groups in the peptide and positively charged side chains surrounding the binding site. In contrast, the final stereospecific complex, which matches the crystal structure, features completely different interactions, namely the burial of the hydrophobic side chain of the peptide C-terminal residue and backbone hydrogen bonds. The simulations show that nonnative salt bridges stabilize kinetically the encounter complex during binding. Unbinding follows the inverse sequence of events with the same nonnative salt bridges in the encounter complex. Thus, in contrast to protein folding, which is driven by native interactions, the binding of charged peptides can be steered by nonnative interactions, which might be a general mechanism, e.g., in the recognition of histone tails by bromodomains.     

Physical Organization of DNA by Multiple Non-Specific DNA-Binding Modes of Integration Host Factor (IHF)

The integration host factor (IHF) is an abundant nucleoid-associated protein and an essential co-factor for phage λ site-specific recombination and gene regulation in E. coli. Introduction of a sharp DNA kink at specific cognate sites is critical for these functions. Interestingly, the intracellular concentration of IHF is much higher than the concentration needed for site-specific interactions, suggesting that non-specific binding of IHF to DNA plays a role in the physical organization of bacterial chromatin. However, it is unclear how non-specific DNA association contributes to DNA organization. By using a combination of single DNA manipulation and atomic force microscopy imaging methods, we show here that distinct modes of non-specific DNA binding of IHF result in complex global DNA conformations. Changes in KCl and IHF concentrations, as well as tension applied to DNA, dramatically influence the degree of DNA-bending. In addition, IHF can crosslink DNA into a highly compact DNA meshwork that is observed in the presence of magnesium at low concentration of monovalent ions and high IHF-DNA stoichiometries. Our findings provide important insights into how IHF contributes to bacterial chromatin organization, gene regulation, and biofilm formation.     

Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL

To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants. The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination. It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain. This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner.     

Indirect recognition in sequence-specific DNA binding by Escherichia coli integration host factor: the role of DNA deformation energy

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.     

In Vitro Selection of Integration Host Factor Binding Sites

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.     

Structural analysis of cation-pi interactions in DNA binding proteins

Cation-pi interactions play an important role in the stability of protein structures. In this work, we have analyzed the influence of cation-pi interactions in DNA binding proteins. We observed cation-pi interactions in 45 out of 62 DNA binding proteins and there is no significant correlation between the number of amino acid residues and number of cation-pi interactions. These interactions are mainly formed by long-range contacts, and the role of short and medium-range contacts is minimal. The preference of Arg is higher than Lys to form cation-pi interactions. The pair-wise cation-pi interaction energy between aromatic and positively charged residues shows that Arg-Tyr energy is the strongest among the possible six pairs. The structural analysis of cation-pi interaction forming residues shows that Lys, Trp, and Tyr prefer to be in the binding site of protein-DNA complexes. Further, the accessible surface areas of cation-pi interaction forming cationic residues are significantly less than that of other residues. The preference of cation-pi interaction forming residues in different secondary structures shows that Lys prefers to be in strand and Phe prefers to be in turn regions. The results obtained in the present study will be useful in understanding the contribution of cation-pi interactions to the stability and specificity of protein-DNA complexes.     

Modeling DNA deformations induced by minor groove binding proteins

Molecular modeling is used to demonstrate that the major structural deformations of DNA caused by four different minor groove binding proteins, TBP, SRY, LEF-1, and PurR, can all be mimicked by stretching the double helix between two 3'-phosphate groups flanking the binding region. This deformation reproduces the widening of the minor groove and the overall bending and unwinding of DNA caused by protein binding. It also reproduces the principal kinks associated with partially intercalated amino acid side chains, observed with such interactions. In addition, when protein binding involves a local transition to an A-like conformation, phosphate neutralization, via the formation of protein-DNA salt bridges, appears to favor the resulting deformation.