Design, synthesis and in vitro evaluation of novel bivalent S-adenosylmethionine analogues

In optimal cases, bivalent ligands can bind with exceptionally high affinity to their protein targets. However, designing optimised linkers, that orient the two binding groups perfectly, is challenging, and yet crucial in both fragment-based ligand design and in the discovery of bisubstrate enzyme inhibitors. To further our understanding of linker design, a series of novel bivalent S-adenosylmethionine (SAM) analogues were designed with the aim of interacting with the MetJ dimer in a bivalent sense (1:1 ligand/MetJ dimer). A range of ligands was synthesised and analyzed for ability to promote binding of the Escherichia coli methionine repressor, MetJ, to its operator DNA. Binding of bivalent SAM analogues to the MetJ homodimer in the presence of operator DNA was evaluated by fluorescence anisotropy and the effect of linker length and structure was investigated. The most effective bivalent ligand identified had a flexible linker, and promoted the DNA-protein interaction at 21-times lower concentration than the corresponding monovalent control compound.     

Structural basis for the differential regulation of DNA by the methionine repressor MetJ

The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.     

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.     

Probing the molecular mechanism of action of co-repressor in the E. coli methionine repressor-operator complex using surface plasmon resonance (SPR).

We have studied quantitatively the effect of the corepressor, S-adenosylmethionine (SAM), on the interaction between the E. coli methionine repressor, MetJ, and an idealised operator fragment, by recording measurements of surface plasmon resonance using a BIAcore instrument. We have recorded kinetic binding data in the presence of SAM, which carries a net positive charge, and two corepressor analogues, adenosylornithine (AO) and aza-SAM, which differ in the location of the atom carrying the positive charge. Our data support the hypothesis that the effect of the corepressor is electrostatic in origin. The difference in electrostatic interaction energy between the SAM- and AO-repressor-operator complexes of approximately 3.5 kJ/mol calculated from the known three-dimensional structure is within the range of our experimentally determined values of 2.8-4.3 kJ/mol. These results illustrate the potential of SPR measurements for studying protein-nucleic acid interactions.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae

Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p. In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity. We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar. However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p. The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process. Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding. Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p. Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.     

Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli.

The thermal stability of the methionine repressor protein from Escherichia coli (MetJ) has been examined over a wide range of pH (pH 3.5-10) and ionic strength conditions using differential scanning calorimetry. Under reducing conditions, the transitions are fully reversible, and thermograms are characteristic of the cooperative unfolding of a globular protein with a molecular weight corresponding to the MetJ dimer, indicating that no dissociation of this dimeric protein occurs before unfolding of the polypeptide chains under most conditions. In the absence of reducing agent, repeated scans in the calorimeter show only partial reversibility, though the thermodynamic parameters derived from the first scans are comparable to those obtained under fully reversible conditions. The protein is maximally stable (Tm 58.5 degrees C) at about pH 6, close to the estimated isoelectric point, and stability is enhanced by increasing ionic strength in the range I = 0.01-0.4 M. The average calorimetric transition enthalpy (delta Hm) for the dimer is 505 +/- 28 kJ mol-1 under physiological conditions (pH 7, I = 0.125, Tm = 53.2 degrees C) and shows a small temperature dependence which is consistent with an apparent denaturational heat capacity change (delta Cp) of about +8.9 kJ K-1 mol-1. The effects of both pH and ionic strength on the transition temperature and free energy of MetJ unfolding are inconsistent with any single amino acid contribution and are more likely the result of more general electrostatic interactions, possibly including significant contributions from electrostatic repulsion between the like-charged monomers which can be modeled by a Debye-Hückel screened potential.     

Identification of the SSB Binding Site on E. coli RecQ Reveals a Conserved Surface for Binding SSB's C Terminus

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.     

DNA binding and bending by HMG boxes: energetic determinants of specificity

To clarify the physical basis of DNA binding specificity, the thermodynamic properties and DNA binding and bending abilities of the DNA binding domains (DBDs) of sequence-specific (SS) and non-sequence-specific (NSS) HMG box proteins were studied with various DNA recognition sequences using micro-calorimetric and optical methods. Temperature-induced unfolding of the free DBDs showed that their structure does not represent a single cooperative unit but is subdivided into two (in the case of NSS DBDs) or three (in the case of SS DBDs) sub-domains, which differ in stability. Both types of HMG box, most particularly SS, are partially unfolded even at room temperature but association with DNA results in stabilization and cooperation of all the sub-domains. Binding and bending measurements using fluorescence spectroscopy over a range of ionic strengths, combined with calorimetric data, allowed separation of the electrostatic and non-electrostatic components of the Gibbs energies of DNA binding, yielding their enthalpic and entropic terms and an estimate of their contributions to DNA binding and bending. In all cases electrostatic interactions dominate non-electrostatic in the association of a DBD with DNA. The main difference between SS and NSS complexes is that SS are formed with an enthalpy close to zero and a negative heat capacity effect, while NSS are formed with a very positive enthalpy and a positive heat capacity effect. This indicates that formation of SS HMG box-DNA complexes is specified by extensive van der Waals contacts between apolar groups, i.e. a more tightly packed interface forms than in NSS complexes. The other principal difference is that DNA bending by the NSS DBDs is driven almost entirely by the electrostatic component of the binding energy, while DNA bending by SS DBDs is driven mainly by the non-electrostatic component. The basic extensions of both categories of HMG box play a similar role in DNA binding and bending, making solely electrostatic interactions with the DNA.     

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ~10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.     

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.