Krystyna Zakrzewska

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.

JUMNA (junction minimisation of nucleic acids)

The latest version of JUMNA program for the energy optimisation of nucleic acids and nucleic acid-ligand complexes is described. The force field used, input and output data, various options for symmetry, conformational constraints and energy mapping are discussed as well as recent combinatorial search techniques.

CURVES+ web server for analyzing and visualizing the helical, backbone and groove parameters of nucleic acid structures

Curves+, a revised version of the Curves software for analyzing the conformation of nucleic acid structures, is now available as a web server. This version, which can be freely accessed at http://gbio-pbil.ibcp.fr/cgi/Curves_plus/, allows the user to upload a nucleic acid structure file, choose the nucleotides to be analyzed and after optionally setting a number of input variables, view the numerical and graphic results online or download files containing a set of helical, backbone and groove parameters that fully describe the structure. PDB format files are also provided for offline visualization of the helical axis and groove geometry.

μABC: a systematic microsecond molecular dynamics study of tetranucleotide sequence effects in B-DNA.

We present the results of microsecond molecular dynamics simulations carried out by the ABC group of laboratories on a set of B-DNA oligomers containing the 136 distinct tetranucleotide base sequences. We demonstrate that the resulting trajectories have extensively sampled the conformational space accessible to B-DNA at room temperature. We confirm that base sequence effects depend strongly not only on the specific base pair step, but also on the specific base pairs that flank each step. Beyond sequence effects on average helical parameters and conformational fluctuations, we also identify tetranucleotide sequences that oscillate between several distinct conformational substates. By analyzing the conformation of the phosphodiester backbones, it is possible to understand for which sequences these substates will arise, and what impact they will have on specific helical parameters.

Local and global effects of strong DNA bending induced during molecular dynamics simulations

DNA bending plays an important role in many biological processes, but its molecular and energetic details as a function of base sequence remain to be fully understood. Using a recently developed restraint, we have studied the controlled bending of four different B-DNA oligomers using molecular dynamics simulations. Umbrella sampling with the AMBER program and the recent parmbsc0 force field yield free energy curves for bending. Bending 15-base pair oligomers by 90° requires roughly 5 kcal mol−1, while reaching 150° requires of the order of 12 kcal mol−1. Moderate bending occurs mainly through coupled base pair step rolls. Strong bending generally leads to local kinks. The kinks we observe all involve two consecutive base pair steps, with disruption of the central base pair (termed Type II kinks in earlier work). A detailed analysis of each oligomer shows that the free energy of bending only varies quadratically with the bending angle for moderate bending. Beyond this point, in agreement with recent experiments, the variation becomes linear. An harmonic analysis of each base step yields force constants that not only vary with sequence, but also with the degree of bending. Both these observations suggest that DNA is mechanically more complex than simple elastic rod models would imply.

Multistep Drug Intercalation: Molecular Dynamics and Free Energy Studies of the Binding of Daunomycin to DNA

Atomic-scale molecular dynamics and free energy calculations in explicit aqueous solvent are used to study the complex mechanism by which a molecule can intercalate between successive base pairs of the DNA double helix. We have analyzed the intercalation pathway for the anticancer drug daunomycin using two different methods: metadynamics and umbrella sampling. The resulting free energy pathways are found to be consistent with one another and point, within an equilibrium free energy context, to a three-step process. Daunomycin initially binds in the minor groove of DNA. An activated step then leads to rotation of the drug, coupled with DNA deformation that opens a wedge between the base pairs, bends DNA toward the major groove, and forms a metastable intermediate that resembles structures seen within the interfaces between DNA and minor-groove-binding proteins. Finally, crossing a small free energy barrier leads to further rotation of daunomycin and full intercalation of the drug, reestablishing stacking with the flanking base pairs and straightening the double helix.

Calculation and analysis of low frequency normal modes for DNA

Normal mode calculations for two alternating sequence dodecamers in A, B, and Z conformations have been performed in dihedral angle space extended to endocyclic valence angles to account for sugar ring flexibility. Normal modes are analyzed in terms of helicoidal and backbone parameter variations with special attention being paid to global deformations of the double helix such as bending, twisting, or stretching. Results show that the allomorphic form of DNA has the largest influence on the flexibility of the sugar‐phosphate backbone. Amplitudes of these vibrations follow the order: B > Z > A. In contrast, the amplitudes of helicoidal parameter variations are much more dependent on the base sequence. Global deformations of the double helix occur with characteristic times in the range of 1 to 10 ps and can be of mixed character, the strongest bending mode being at the same the time strongest stretching mode.

Analyzing ion distributions around DNA

We present a new method for analyzing ion, or molecule, distributions around helical nucleic acids and illustrate the approach by analyzing data derived from molecular dynamics simulations. The analysis is based on the use of curvilinear helicoidal coordinates and leads to highly localized ion densities compared to those obtained by simply superposing molecular dynamics snapshots in Cartesian space. The results identify highly populated and sequence-dependent regions where ions strongly interact with the nucleic and are coupled to its conformational fluctuations. The data from this approach is presented as ion populations or ion densities (in units of molarity) and can be analyzed in radial, angular and longitudinal coordinates using 1D or 2D graphics. It is also possible to regenerate 3D densities in Cartesian space. This approach makes it easy to understand and compare ion distributions and also allows the calculation of average ion populations in any desired zone surrounding a nucleic acid without requiring references to its constituent atoms. The method is illustrated using microsecond molecular dynamics simulations for two different DNA oligomers in the presence of 0.15 M potassium chloride. We discuss the results in terms of convergence, sequence-specific ion binding and coupling with DNA conformation.

Protein–DNA Recognition Triggered by a DNA Conformational Switch

How DNA-binding proteins find their target sites remains a fascinating question. Early work on the Lac repressor showed that proteins specifically bind faster than simple diffusion allows. This led to the idea that recognition could be accelerated by combining diffusion with sliding along DNA and hopping between neighboring strands. This, in turn, implied that proteins could interact with DNA in a distinct nonspecific manner. Experimental work has confirmed that proteins can indeed slide along DNA, although typical sliding distances vary from protein to protein. Recent work also indicates that sliding follows the helical grooves of DNA. Crystallography of proteins bound to noncognate sites, NMR spectroscopy, and molecular simulations have all provided data on nonspecific binding, notably suggesting that proteins maintain similar orientations with respect to DNA in nonspecifically and specifically bound states (see, e.g., Refs. [7–9]). However, little is known about the transition between these states, although theoretical studies have suggested that a switching mechanism may exist, possibly involving a protein conformational change. To analyze this problem at the atomic level, we carried out a molecular dynamics (MD) study on the dissociation of a specific protein–DNA complex in water, starting from the bound conformation. We studied the sex-determining region Y (SRY) protein, which affects the gender selection in mammals and is linked to a number of gender-related pathologies. The SRY protein binds in the minor groove of DNA, optimally at an (A/T)AACAAT sequence, and opens the minor groove by partial intercalation of an isoleucine residue (Ile13) between two adjacent AT base pairs and induces local unwinding and bending of DNA away from the protein. Using a specially designed restraint for the minimal atomic distance between any pair of nonhydrogen atoms across the protein–DNA interface (dMIN), we were able to control the dissociation of the SRY protein from a 14-base-pair (bp) DNA oligomer (5’-CCTGCACAAACACC-3’) without biasing the conformational pathway. Using this approach, roughly 0.6 ms of umbrella sampling led to a free-energy profile for the dissociation/association process. This profile showed a free-energy gain of 11.5 kcal mol 1 because of the SRY-DNA binding; this binding process includes passage of an energy barrier of roughly 4 kcalmol 1 at a separation of 4.2–3.5 and a secondary barrier of 2 kcalmol 1 at 3.1 (see Figure S1 in the Supporting Information). We investigated the conformational aspects of this pathway to understand the recognition mechanism. An initial analysis showed that the conformation of the SRY protein remained remarkably stable during its separation from the DNA. Although the Nand C-terminal tails were very flexible, the three-a-helix protein core varied by a root-mean-square deviation (RMSD) of 2.2 at most (see Figure S1 in the Supporting Information). In contrast, the DNA oligomer underwent considerable change, linked to the SRY-induced deformations. However, our initial analysis of the separation profile also showed that many DNA conformations and protein locations occurred for a single minimal pair distance along the separation pathway. We reduced this problem by using the distance dAXC from the center of the DNA helical axis to the center of mass of the core region of the SRY protein. This distance varies almost monotonically with the minimal pair distance for dMIN 20 . The most interesting feature lies between these two regions (13 > dAXC> 20 ), where the DNA conformations clearly split into two paths (green and red). The center of the two-path region occurs around dAXC= 16 . Here, both paths are found at a RMSD value of around 3 relative to the bound DNA reference state, whereas the upper path (path 1) is located at a RMSD value of 5.2 relative to the unbound [*] Dr. B. Bouvier, Dr. K. Zakrzewska, Dr. R. Lavery Universit Lyon 1/CNRS, UMR 5086, Bases Mol culaires et Structurales des Syst mes Infectieux, IBCP 7 passage du Vercors, 69367 Lyon (France) E-mail: richard.lavery@ibcp.fr Homepage: http://www.ibcp.fr

A Theoretical Study of the Sequence Specificity in Binding of Lexitropsins to B-DNA

A theoretical study is presented on the binding to B-DNA of a series of lexitropsins, these ligands being netropsin derivatives in which one or both of the pyrrole rings have been replaced by imidazoles. The best complexes have been located by energy minimisation taking into account nucleic acid flexibility, ligand flexibility, explicit, mobile counterions and solvent dielectric effects. Calculations have been performed for two homopolymeric DNA receptor sequences, AT base sequence, which only decreases in the imidazole derivatives. These results emphasize the decisive role of the molecular electrostatic potential of the nucleic acid in determining the sequence selectivity of these ligands, as opposed to the postulated role of adenine C2 - pyrrole beta hydrogen contacts.