Topoisomerase IV bends and overtwists DNA upon binding

Escherichia coli topoisomerase IV (Topo IV) is an essential ATP-dependent enzyme that unlinks sister chromosomes during replication and efficiently removes positive but not negative supercoils. In this article, we investigate the binding properties of Topo IV onto DNA in the absence of ATP using a single molecule micromanipulation setup. We find that the enzyme binds cooperatively (Hill coefficient alpha approximately 4) with supercoiled DNA, suggesting that the Topo IV subunits assemble upon binding onto DNA. It interacts preferentially with (+) rather than (-) supercoiled DNA (Kd+=0.15 nM, Kd-=0.23 nM) and more than two orders-of-magnitude more weakly with relaxed DNA (Kd0 approximately 36 nM). Like gyrase but unlike the eukaryotic Topo II, Topo IV bends DNA with a radius 0= 6.4 nm and locally changes its twist and/or its writhe by 0.16 turn per bound complex. We estimate its free energy of binding and study the dynamics of interaction of Topo IV with DNA at the binding threshold. We find that the protein/DNA complex alternates between two states: a weakly bound state where it stays with probability p = 0.89 and a strongly bound state (with probability p = 0.11). The methodology introduced here to characterize the Topo IV/DNA complex is very general and could be used to study other DNA/protein complexes.     

What determines the van der Waals coefficient beta in the LIE (linear interaction energy) method to estimate binding free energies using molecular dynamics simulations?

Recently a semiempirical method has been proposed by Aqvist et al. to calculate absolute and relative binding free energies. In this method, the absolute binding free energy of a ligand is estimated as deltaGbind = alpha + beta, where Vel(bound) and Vvdw(bound) are the electrostatic and van der Waals interaction energies between the ligand and the solvated protein from an molecular dynamics (MD) trajectory with ligand bound to protein and Vel(free) and Vel(free) and Vvdw(free) are the electrostatic and van der Waals interaction energies between the ligand and the water from an MD trajectory with the ligand in water. A set of values, alpha = 0.5 and beta = 0.16, was found to give results in good agreement with experimental data. Later, however, different optimal values of beta were found in studies of compounds binding to P450cam and avidin. The present work investigates how the optimal value of beta depends on the nature of binding sites for different protein-ligand interactions. By examining seven ligands interacting with five proteins, we have discovered a linear correlation between the value of beta and the weighted non-polar desolvation ratio (WNDR), with a correlation coefficient of 0.96. We have also examined the ability of this correlation to predict optimal values of beta for different ligands binding to a single protein. We studied twelve neutral compounds bound to avidin. In this case, the WNDR approach gave a better estimate of the absolute binding free energies than results obtained using the fixed value of beta found for biotin-avidin. In terms of reproducing the relative binding free energy to biotin, the fixed-beta value gave better results for compounds similar to biotin, but for compounds less similar to biotin, the WNDR approach led to better relative binding free energies.     

Thermodynamics calculation of protein–ligand interactions by QM/MM polarizable charge parameters

The calculation of protein–ligand binding free energy (ΔG) is of great importance for virtual screening and drug design. Molecular dynamics (MD) simulation has been an attractive tool to investigate this scientific problem. However, the reliability of such approach is affected by many factors including electrostatic interaction calculation. Here, we present a practical protocol using quantum mechanics/molecular mechanics (QM/MM) calculations to generate polarizable QM protein charge (QMPC). The calculated QMPC of some atoms in binding pockets was obviously different from that calculated by AMBER ff03, which might significantly affect the calculated ΔG. To evaluate the effect, the MD simulations and MM/GBSA calculation with QMPC for 10 protein–ligand complexes, and the simulation results were then compared to those with the AMBER ff03 force field and experimental results. The correlation coefficient between the calculated ΔΔG using MM/GBSA under QMPC and the experimental data is .92, while that with AMBER ff03 force field is .47 for the complexes formed by streptavidin or its mutants and biotin. Moreover, the calculated ΔΔG with QMPC for the complexes formed by ERβ and five ligands is positively related to experimental result with correlation coefficient of .61, while that with AMBER ff03 charge is negatively related to experimental data with correlation coefficient of .42. The detailed analysis shows that the electrostatic polarization introduced by QMPC affects the electrostatic contribution to the binding affinity and thus, leads to better correlation with experimental data. Therefore, this approach should be useful to virtual screening and drug design.     

Tailoring the activity of restriction endonuclease PleI by PNA-induced DNA looping

DNA looping is one of the key factors allowing proteins bound to different DNA sites to signal one another via direct contacts. We demonstrate that DNA looping can be generated in an arbitrary chosen site by sequence-directed targeting of double-stranded DNA with pseudocomplementary peptide-nucleic acids (pcPNAs). We designed pcPNAs to mask the DNA from cleavage by type IIs restriction enzyme PleI while not preventing the enzyme from binding to its primary DNA recognition site. Direct interaction between two protein molecules (one bound to the original recognition site and the other to a sequence-degenerated site) results in a totally new activity of PleI: it produces a nick near the degenerate site. The PNA-induced nicking efficiency varies with the distance between the two protein-binding sites in a phase with the DNA helical periodicity. Our findings imply a general approach for the fine-tuning of proteins bound to DNA sites well separated along the DNA chain.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

Theoretical prediction of the binding free energy for mutants of replication protein A

The replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), single stranded DNA (ssDNA) binding protein required for pivotal functions in the cell metabolism, such as chromosomal replication, prevention of hairpin formation, DNA repair and recombination, and signaling after DNA damage. Studies based on deletions and mutations have identified the high affinity ssDNA binding domains in the 70 kDa subunit of RPA, regions A and B. Individually, the domain A and B have a low affinity for ssDNA, while tandems composed of AA, AB, BB, and BA sequences bind the ssDNA with moderate to high affinity. Single and double point mutations on polar residues in the binding domains leads to a reduction in affinity of RPA for ssDNA, in particular when two hydrophilic residues are involved. In view of these results, we performed a study based on molecular dynamics simulation aimed to reproduce the experimental change in binding free energy, ΔΔG, of RPA70 mutants to further elucidate the nature of the protein-ssDNA interaction. The MM-PB(GB)SA methods implemented in Amber10 and the code FoldX were used to estimate the binding free energy. The theoretical and experimental ΔΔG values correlate better when the results are obtained by MM-PBSA calculated on individual trajectories for each mutant. In these conditions, the correlation coefficient between experimental and theoretical ΔΔG reaches a value of 0.95 despite the overestimation of the energy change by one order of magnitude. The decomposition of the MM-GBSA energy per residue allows us to correlate the change of the affinity with the residue polarity and energy contribution to the binding. The method revealed reliable predictions of the change in the affinity in function of mutations, and can be used to identify new mutants with distinct binding properties.     

The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.     

Diffusion of the Restriction Nuclease EcoRI along DNA

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 × 10⁴ bp² s^− 1 EcoRI is able to diffuse ∼ 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.     

The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.     

Radiation affects binding of Fpg repair protein to an abasic site containing DNA

During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site. Is this step affected by exposure to ionizing radiation? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (P1G-LlFpg) lacking strand cleavage activity. Upon irradiation of the complex, the ratio of bound/free partners decreased. When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA. Thus irradiation hinders Fpg-DNA binding because of the damage to the protein. Using our radiolytic attack simulation procedure RADACK (Begusova et al., J. Biomol. Struct. Dyn. 19, 141-157, 2001), we reveal the potential hot spots for damage in the irradiated protein. Most of them are essential for the interaction of Fpg with DNA, which explains the radiation-induced loss of binding ability of Fpg. The doses necessary to destroy the complex are higher than those inactivating Fpg irradiated separately. As confirmed by our calculations, this can be explained by the partial protection of the protein by the bound DNA.     

Simulating the effect of DNA polymerase mutations on transition-state energetics and fidelity: evaluating amino acid group contribution and allosteric coupling for ionized residues in human pol beta

The control of the catalytic power and fidelity of DNA polymerases involves the complex combined effect of the protein residues, the Mg2+ ions, and the interaction between the DNA bases. In an attempt to advance the understanding of catalytic control, we analyze the effect of the protein residues, taking human DNA polymerase beta as a model system. Specifically, we examine the ability of different theoretical models to reproduce the effect of ionized residues on the transition state (TS) binding energy and the corresponding k(pol)/KD. We also explore the role of the Mg2+ ions in the binding and catalysis processes. The application of the microscopic linear response approximation (LRA) and the semimacroscopic PDLD/S-LRA methods to a benchmark of mutational studies produces a semiquantitative correlation and indicates that these methods can provide predictive power. However, pre-steady-state and steady-state kinetic studies currently available do not give a unique benchmark, owing principally to widely varying experimental conditions. We believe that a more uniform experimental benchmark is needed for further refinement of the theoretical models. The analysis of the correlation between the results obtained by a rigorous thermodynamic cycle and by simpler approximations indicates that the protein reorganization between the open, i.e., unbound, form and the closed form does not change the magnitude of the calculated mutational effects in a major way for the experimental data used in this study. The use of the PDLD/S-LRA group contributions allows us to construct energy-based correlation diagrams that can help toward understanding the coupling, i.e., transfer of information, between the base-binding and catalytic sites and to gain a deeper insight into the molecular basis of DNA replication fidelity. Our analysis suggests that the allosteric matrix obtained by subtracting the correlation matrix of the correct and incorrect base pairs should prove useful in exploring the information transfer occurring between the base-binding and catalytic sites. This type of treatment should be especially effective when coupled with structural studies of polymerase-DNA-base mispair ternary complexes and studies using polymerase double mutants. We discuss the potential of direct calculations of binding energy of the TS in a rational design of TS analogues and in drug design.     

DNA phosphate crowding correlates with protein cationic side chain density and helical curvature in protein/DNA crystal structures

Sequence-specific binding of proteins to their DNA targets involves a complex spectrum of processes that often induce DNA conformational variation in the bound complex. The forces imposed by protein binding that cause the helical deformations are intimately interrelated and difficult to parse or rank in importance. To investigate the role of electrostatics in helical deformation, we quantified the relationship between protein cationic residue density (Cpc) and DNA phosphate crowding (Cpp). The correlation between Cpc and Cpp was then calculated for a subset of 58 high resolution protein–DNA crystal structures. Those structures containing strong Cpc/Cpp correlation (>±0.25) were likely to contain DNA helical curvature. Further, the correlation factor sign predicted the direction of helical curvature with positive (16 structures) and negative (seven structures) correlation containing concave (DNA curved toward protein) and convex (DNA curved away from protein) curvature, respectively. Protein–DNA complexes without significant Cpc/Cpp (36 structures) correlation (-0.25<0<0.25) tended to contain DNA without significant curvature. Interestingly, concave and convex complexes also include more arginine and lysine phosphate contacts, respectively, whereas linear complexes included essentially equivalent numbers of Lys/Arg phosphate contacts. Together, these findings suggest an important role for electrostatic interactions in protein–DNA complexes involving helical curvature.     

The study of DNA sequences by their sequences of twist angles

To study the properties of DNA sequences we have transformed the sequences of bases into the sequences of twist angles along the chain of DNA double helix by using the Dickerson sum function. The Fourier transform and the auto-correlation function of the twist angles sequences have been used to study the periodicity and randomness of the original DNA sequences. Basing on the correlation coefficient, a "distance" between two DNA fragments has been defined and used to compare some realistic DNA sequences. It is hoped that the techniques developed here could be used to analyze more realistic DNA sequences.     

Conserved DNA structures in origins of replication.

According to the model of Bramhill and Kornberg, initiation of DNA replication in prokaryotes involves binding of an initiator protein to origin DNA and subsequent duplex opening of adjacent direct repeat sequences. In this report, we have used computer analysis to examine the higher-order DNA structure of a variety of origins of replication from plasmids, phages, and bacteria in order to determine whether these sequences are localized in domains of altered structure. The results demonstrate that the primary sites of initiator protein binding lie in discrete domains of DNA bending, while the direct repeats lie within well-defined boundaries of an unusual anti-bent domain. The anti-bent structures arise from a periodicity of A3 and T3 tracts which avoids the 10-11 bp bending periodicity. Since DNA fragments which serve as replicators in yeast also contain these two conserved structural elements, the results provide new insight into the universal role of conserved DNA structures in DNA replication.     

Membrane-bound structure and energetics of alpha-synuclein

Aggregation and fibrillation of alpha-synuclein bound to membranes are believed to be involved in Parkinson's and other neurodegenerative diseases. On SDS micelles, the N-terminus of alpha-synuclein forms two curved helices linked by a short loop. However, its structure on lipid bilayers has not been experimentally resolved. Using MD simulations with an implicit membrane model we show here that, on a planar mixed membrane, the truncated alpha-synuclein (residues 1-95) forms a bent helix. Bending of the helix is not due to the protein sequence or membrane binding, but to collective motions of the long helix. The backbone of the helix is approximately 2.5 A above the membrane surface, with some residues partially inserted in the membrane core. The helix periodicity is 11/3 (11 residues complete three full turns) as opposed to 18/5 periodicity of an ideal alpha-helix, with hydrophobic residues towards the membrane, negatively charged residues towards the solvent and lysines on the polar/nonpolar interface. A series of threonines, which are characteristic for alpha-synuclein and perhaps a phosphorylation site, is also located at the hydrophobic/hydrophilic interface with their side chain often hydrogen bonded to the main-chain atom. The calculations show that the energy penalty for change in periodicity from the 18/5 to 11/3 on the anionic membrane is overcome by favorable solvation energy. The binding of truncated alpha-synuclein to membranes is weak. It prefers anionic membranes but it also binds marginally to a neutral membrane, via its C-terminus. Dimerization of helical monomers on the mixed membrane is energetically favorable. However, it slightly interferes with membrane binding. This might promote lateral diffusion of the protein on the membrane surface and facilitate assembly of oligomers that precede fibrillation.