A model for the LexA repressor DNA complex

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein–DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two β-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed. © 1995 Wiley-Liss, Inc.     

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein‐DNA binding affinities, nitrocellulose filter binding assays with ³²P‐labeled DNA quantify K_d values from 10^?12 to 10^?8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio k_off/k_on determines K_d. However, for lactose repressor protein (LacI) and an engineered repressor protein (“LLhF”) binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below K_d (equilibrium binding regime) must be analyzed differently than those with DNA near or above K_d (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10^?9 M. Although variation occurred among different lots of sensor tips, binding events with K_d ≥ 10^?8 M should reliably be in the equilibrium binding regime. We also observed effects from multi‐valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein‐DNA binding and to assess the affinity of fructose‐1‐kinase for the DNA‐LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.     

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1,2, and 5 positions of the α3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNTTT-. a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12mol/L1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the     

Anatomy of specific interactions between lambda repressor and operator DNA

Recognition of specific DNA sequences by proteins is essential for regulation of gene expression. To fully understand the recognition mechanism, it is necessary to understand not only the structure of the specific protein-DNA interactions but also the energetics. We therefore performed a computer analysis in which a phage DNA-binding protein, lambda repressor, was used to examine the changes in binding free energy (DeltaDeltaG) and its energy components caused by single base mutations. We then determined which of the calculated energy components best correlated with the experimental data. The experimental DeltaDeltaG values were well reproduced by the calculations. Component analysis revealed that the electrostatic and hydrogen bond energies were most strongly correlated with the experimental data. Among the 51 single base-substitution mutants examined, positive DeltaDeltaG values, corresponding to weakened binding, were caused by the loss of favorable electrostatic interactions and hydrogen bonds, the introduction of steric collisions and electrostatic repulsion, the loss of favorable interactions with a thymine methyl group, and the increase of unfavorable hydration energy from isolated DNA. This analysis also showed distinct patterns of recognition at A-T and G-C positions, as different combinations of energy components were involved in DeltaDeltaG caused by the two substitution types. We have thus been able to identify the energy components that most strongly correlate with sequence-dependent DeltaDeltaG and determine their contribution to the specificity of DNA sequence recognition by the lambda repressor. Application of this method to other systems should provide additional insight into the molecular mechanism of protein-DNA recognition.     

The Role of Duplex Ends in the Spontaneous Interaction of Homologous Linear DNA Fragments

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA–DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.     

The HhH domain of the human DNA repair protein XPF forms stable homodimers

The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.     

New and old roles of the double-stranded RNA-binding domain

RNA-binding proteins can strongly regulate and influence the cellular function and fate of an RNA molecule. Of the many described nucleic acid-binding domains, the double-stranded RNA-binding domain (dsRBD) is a highly specialized example found in a wide variety of proteins with diverse cellular functions. Mostly present in multiple copies and highly homologous to one another, the individual functional specificity of dsRBDs is now becoming apparent. Here we review recent evidence showing that single dsRBDs within individual proteins are capable of distinct in vivo functions. Not only does this enable dsRBD-containing proteins to increase their functional diversity but it also reveals novel and unexpected roles that dsRBDs can perform.     

Operator recognition by the phage 434 cI repressor: MD simulations of free and bound 50-bp DNA reveal important differences between the OR1 and OR2 sites

Using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp DNA sequence containing the 434 bacteriophage operators OR1 and OR2 separated by an 8-bp spacer. Two simulations of 1 ns each were carried out, with DNA alone and with DNA complexed to dimers of the R1-69 DNA binding domain of the phage 434 cI repressor protein at the OR1 and OR2 sites. Strong correlations among average structural parameters are observed between our simulations and available experimental data for the bound OR1/OR2 subsites. In the free state, some differences appear between the three relevant fragments (OR1, the spacer, and OR2). Unbound OR1 exhibits a large, shallow major groove into which the base atoms protrude and is also bent toward the major groove. This structure is maintained because structural fluctuations are weak. Unbound OR2 resembles canonical B-DNA although the structural parameters show greater fluctuations, essentially due to a malleable step (the innermost CpA/TpG), absent in OR1. Complexation with the proteins slightly alters the base positions but strongly modifies the sugar and backbone motions. The most crucial repressor effects are changes in the flexibility of the OR1/OR2 sites. Structural fluctuations are enhanced for OR1, conferring a favorable energetic contribution to the OR1 binding, whereas they are reduced for OR2. Therefore, both structural and dynamic properties of DNA suggest OR1 is the most attractive site for the repressor, which may explain the different binding association constants observed for the OR1 and OR2 sites. Finally, we also investigated the impact of the protein on the DNA backbone dynamics and find that direct or indirect interactions facilitate the DNA structural variations required for achieving complementarity with the protein.     

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.     

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: A role for N-terminal residues in tandem binding

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of ²H,¹⁵N,¹³C labelled intact and (des2–7) trp repressor (2–7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone ¹HN, ¹⁵N, ¹³C positions in both forms of the protein and for¹³ C in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of 2–7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178–182]. Comparison of the backbone chemical shifts of DNA-bound 2–7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2–12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and 2–7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.     

An allosteric redox switch in domain V of β2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition

β₂-glycoprotein I (β₂GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that β₂GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to β₂GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate β₂GPI''s structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of β₂GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating β₂GPI''s physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.     

Influence of DNA aptamer structure on the specificity of binding to Taq DNA polymerase

The secondary structure of DNA aptamer to Taq DNA polymerase was established as a hairpin. Both stem and loop structures of DNA ligand were shown to be involved in the interaction with Taq DNA polymerase. Moreover, the structure and sequence of DNA aptamer that was the most effective inhibitor of DNA polymerase activity were established. This crucial structure was evaluated as a GC-rich stem longer than 17 bp, and a loop consisting of 12 bases with strictly determined nucleotide sequence. It was demonstrated that nucleotide in position 23 counting from the 5"-end of DNA ligand was involved in direct contact with Taq DNA polymerase. The ability of optimized DNA aptamer TQ21-11 to form a complex with the enzyme was increased 5-fold in comparison to the initial aptamer.     

Lac repressor–operator interaction: N-terminal peptide backbone 1H and 15N chemical shifts upon complex formation with DNA

When the lac repressor tetramer is bound to its DNA operator, methylation protection shows the nearly symmetric operator half-sites are contacted asymmetrically. This asymmetric binding results from the DNA sequence/structure. The reported structure of lac repressor N-terminal fragment and an 11 base-pair operator left half-site provides no information concerning the effect of asymmetric binding, from left operator half-site to right half-site, upon the polypeptide backbone. We isolated uniformly ¹⁵N labeled 56 amino acid wild-type (HP56WT) and 64 residue mutant [Pro3>Tyr3] (HP64tyr3) lac repressor N-terminal DNA binding fragments for ¹H/¹⁵N NMR studies with the left and right operators separately. Spectral coincidence of these longer fragments, indicating structural similarity with a protease derived 51 amino acid fragment for which the amide correlations are assigned, allows for assignment of the common amide resonances. For both HP56WT and HP64tyr3, spectral overlap of the amide correlation peaks reveals the polypeptide backbones of the uncomplexed polypeptides are structurally similar. Likewise the complexes of the peptides to the 11 base-pair lac left operator half-site are similar. On the other hand, complexes of HP56WT and the left compared to the right lac operator half-site show different residues of the polypeptide are affected by binding different half-sites of the operator. Thus, the DNA sequence/structure transmits asymmetry to the polypeptide backbone of the interacting protein.