Minor groove deformability of DNA: a molecular dynamics free energy simulation study

The conformational deformability of nucleic acids can influence their function and recognition by proteins. A class of DNA binding proteins including the TATA box binding protein binds to the DNA minor groove, resulting in an opening of the minor groove and DNA bending toward the major groove. Explicit solvent molecular dynamics simulations in combination with the umbrella sampling approach have been performed to investigate the molecular mechanism of DNA minor groove deformations and the indirect energetic contribution to protein binding. As a reaction coordinate, the distance between backbone segments on opposite strands was used. The resulting deformed structures showed close agreement with experimental DNA structures in complex with minor groove-binding proteins. The calculated free energy of minor groove deformation was approximately 4-6 kcal mol(-1) in the case of a central TATATA sequence. A smaller equilibrium minor groove width and more restricted minor groove mobility was found for the central AAATTT and also a significantly ( approximately 2 times) larger free energy change for opening the minor groove. The helical parameter analysis of trajectories indicates that an easier partial unstacking of a central TA versus AT basepair step is a likely reason for the larger groove flexibility of the central TATATA case.     

Towards an understanding of DNA recognition by the methyl-CpG binding domain 1

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations. As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be -3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

Specific minor groove solvation is a crucial determinant of DNA binding site recognition

The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein''s ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein.     

Binding to the minor groove of the double-strand, tau protein prevents DNA from damage by peroxidation

Tau, an important microtubule associated protein, has been found to bind to DNA, and to be localized in the nuclei of both neurons and some non-neuronal cells. Here, using electrophoretic mobility shifting assay (EMSA) in the presence of DNA with different chain-lengths, we observed that tau protein favored binding to a 13 bp or a longer polynucleotide. The results from atomic force microscopy also showed that tau protein preferred a 13 bp polynucleotide to a 12 bp or shorter polynucleotide. In a competitive assay, a minor groove binder distamycin A was able to replace the bound tau from the DNA double helix, indicating that tau protein binds to the minor groove. Tau protein was able to protect the double-strand from digestion in the presence of DNase I that was bound to the minor groove. On the other hand, a major groove binder methyl green as a negative competitor exhibited little effect on the retardation of tau-DNA complex in EMSA. This further indicates the DNA minor groove as the binding site for tau protein. EMSA with truncated tau proteins showed that both the proline-rich domain (PRD) and the microtubule-binding domain (MTBD) contributed to the interaction with DNA; that is to say, both PRD and MTBD bound to the minor groove of DNA and bent the double-strand, as observed by electron microscopy. To investigate whether tau protein is able to prevent DNA from the impairment by hydroxyl free radical, the chemiluminescence emitted by the phen-Cu/H(2)O(2)/ascorbate was measured. The emission intensity of the luminescence was markedly decreased when tau protein was present, suggesting a significant protection of DNA from the damage in the presence of hydroxyl free radical.     

What drives proteins into the major or minor grooves of DNA?

The energetic profiles of a significant number of protein-DNA systems at 20 °C reveal that, despite comparable Gibbs free energies, association with the major groove is primarily an enthalpy-driven process, whereas binding to the minor groove is characterized by an unfavorable enthalpy that is compensated by favorable entropic contributions. These distinct energetic signatures for major versus minor groove binding are irrespective of the magnitude of DNA bending and/or the extent of binding-induced protein refolding. The primary determinants of their different energetic profiles appear to be the distinct hydration properties of the major and minor grooves; namely, that the water in the A+T-rich minor groove is in a highly ordered state and its removal results in a substantial positive contribution to the binding entropy. Since the entropic forces driving protein binding into the minor groove are a consequence of displacing water ordered by the regular arrangement of polar contacts, they cannot be regarded as hydrophobic.     

Distamycin-induced inhibition of formation of a nucleoprotein complex between the terminase small subunit G1P and the non-encapsidated end (pacL site) of Bacillus subtilis bacteriophage SPP1.

The small subunit of the Bacillus subtilis bacteriophage SPP1 terminase (G1P) forms a sequence-specific nucleoprotein complex with the SPP1 non-encapsidated end (pacL site) during initiation of DNA encapsidation. Gel mobility shift assay was used to study the G1P-pacL interaction. Distamycin, a minor groove binder that induces local distortion of the DNA, inhibits G1P-pacL complex formation. The competition of G1P with distamycin for DNA binding at the pacL site is independent of the order of addition of the reactants. Other minor groove binders, such as spermine or Hoechst 33258, which do not distort DNA, failed to compete with G1P for pacL DNA binding. Cationic metals, which generate a repertoire of DNA structures different from that caused by the minor groove binders, can partially reverse the distamycin-induced inhibition of G1P binding to pacL DNA. The major groove binder methyl green, which does not distort sequence-directed bending of pacL DNA, competes with G1P for binding at the pacL site. Our data suggest that the natural sequence-directed bend that exists within the pacL site is the architectural element that facilitates assembly of a nucleoprotein complex and hence initiation of DNA encapsidation by bacteriophage SPP1.     

A new protein domain for binding to DNA through the minor groove.

Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.     

DNA recognition by β-sheets

The modes of DNA recognition by β-sheets are analyzed by using the known crystal and solution three-dimensional structures of DNA-protein complexes. Close fitting of the protein surface and the DNA surface determines the binding geometry. Interaction takes place so that essentially the N-to-C direction of the β-strands either follows or crosses the DNA groove. Upon following the major groove a two-stranded antiparallel β-sheet dives into the groove and contacts DNA bases with its convex side facing the DNA, while upon following the minor groove, it binds around the sugar-phosphate backbones, with its opposite concave side shielding the DNA. In order for the β-strands crossing the minor groove to interact with the DNA, the dinucleotide steps need to almost totally helically untwist and roll around major groove. The β-sheet, on the other hand, needs to adopt a concave curvature on the binding surface in the direction that follows the DNA minor groove, and a convex surface in the direction that bridges the sugar-phosphate backbones across the groove. The result is to produce a hyperbolic paraboloidal DNA-binding surface. © 1998 John Wiley & Sons, Inc. Biopoly 44: 335–359, 1997     

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting.

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.     

Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate.

The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the repressor with lactose operator DNA which occurs, as shown by Gilbert et al. (1), along both the major and the minor groove.     

Methylene Blue Binding to DNA with Alternating AT Base Sequence: Minor Groove Binding is Favored over Intercalation

Abstract

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5′-YpR-3′ intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute-solvent interactions, which are assumed to be nearly constant for the compared complexes of MB with DNA, seem to be justified by the results.     

Methylene blue binding to DNA with alternating AT base sequence: minor groove binding is favored over intercalation

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5'-YpR-3' intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute-solvent interactions, which are assumed to be nearly constant for the compared complexes of MB with DNA, seem to be justified by the results.