Alessandra Villa

Magnesium Ion–Water Coordination and Exchange in Biomolecular Simulations

Magnesium ions have an important role in the structure and folding mechanism of ribonucleic acid systems. To properly simulate these biophysical processes, the applied molecular models should reproduce, among other things, the kinetic properties of the ions in water solution. Here, we have studied the kinetics of the binding of magnesium ions with water molecules and nucleic acid systems using molecular dynamics simulation in detail. We have validated the parameters used in biomolecular force fields, such as AMBER and CHARMM, for Mg(2+) ions and also for the biologically relevant ions Na(+), K(+), and Ca(2+) together with three different water models (TIP3P, SPC/E, and TIP5P). The results show that Mg(2+) ions have a slower exchange rate than Na(+), K(+), and Ca(2+) in agreement with the experimental trend, but the simulated value underestimates the experimentally observed Mg(2+)-water exchange rate by several orders of magnitude, irrespective of the force field and water model. A new set of parameters for Mg(2+) was developed to reproduce the experimental kinetic data. This set also leads to better reproduction of structural data than existing models. We have applied the new parameter set to Mg(2+) binding with a monophosphate model system and with the purine riboswitch, add A-riboswitch. In line with the Mg(2+)-water results, the newly developed parameters show a better description of the structure and kinetics of the Mg(2+)-phosphate binding than all other models. The characterization of the ion binding to the riboswitch system shows that the new parameter set does not affect the global structure of the ribonucleic acid system or the number of ions involved in direct or indirect binding. A slight decrease in the number of water-bridged contacts between A-riboswitch and the Mg(2+) ion is observed. The results support the ability of the newly developed parameters to improve the kinetic description of the Mg(2+) and phosphate ions and their applicability in nucleic acid simulation.

Transferability of Nonbonded Interaction Potentials for Coarse-Grained Simulations: Benzene in Water

Methods to parametrize coarse-grained simulation models for molecular fluids frequently either attempt to match the fluid structure (e.g., pair correlation functions) previously obtained with detailed atomistic models or aim at reproducing macroscopically observable thermodynamic properties. In either case, the coarse-grained models are state-point-dependent, and it is unclear to what extent the models obtained at a given state point are transferable, for example, to different compositions in the case of solution mixtures. Usually, it remains unclear as well whether structure-based potentials reproduce macroscopic thermodynamic properties and, vice versa, if thermodynamics-based potentials reproduce microscopic structural properties. In this paper, we use the Kirkwood-Buff theory of solutions in order to link local structural information and thermodynamic properties sampled with structure-based potentials. We investigate benzene/water mixtures at varying concentrations as a model hydrophobic/hydrophilic system and study the transferability of a coarse-grained model that describes the water and benzene molecules as single interaction sites. The coarse-grained model, parametrized at a high aqueous dilution of benzene, reproduces the Kirkwood-Buff integrals of mixtures obtained with the detailed-atomistic model, and it reproduces the change in the benzene chemical potential with composition up to the concentration of thermodynamic instability. The observed transferability of the potential supports the idea that hydrophobic interactions between small molecules are pairwise additive.

Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study.

Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

Summary Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1–3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Elucidating the Relation between Internal Motions and Dihedral Angles in an RNA Hairpin Using Molecular Dynamics

Molecular dynamics simulations were performed to characterize the internal motions of the ribonucleic acid apical stem loop of human hepatitis B virus. The NMR relaxation rates calculated directly from the trajectory are in good agreement with the experiment. Calculated order parameters follow the experimental pattern. Order parameters lower than 0.8 are observed for nucleotides that are weakly hydrogen bonded to their base pair partner, unpaired, or part of the loop. These residues show slow decay of the internal correlation functions of their base and sugar C-H vectors. Concerted motions around backbone dihedral angles influence the amplitude of motion of the sugar and base C-H vectors. The order parameters for base C-H vectors are also affected by the fluctuation of the glycosidic dihedral angle.

LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures

The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.

Sequence dependency of canonical base pair opening in the DNA double helix

The flipping-out of a DNA base from the double helical structure is a key step of many cellular processes, such as DNA replication, modification and repair. Base pair opening is the first step of base flipping and the exact mechanism is still not well understood. We investigate sequence effects on base pair opening using extensive classical molecular dynamics simulations targeting the opening of 11 different canonical base pairs in two DNA sequences. Two popular biomolecular force fields are applied. To enhance sampling and calculate free energies, we bias the simulation along a simple distance coordinate using a newly developed adaptive sampling algorithm. The simulation is guided back and forth along the coordinate, allowing for multiple opening pathways. We compare the calculated free energies with those from an NMR study and check assumptions of the model used for interpreting the NMR data. Our results further show that the neighboring sequence is an important factor for the opening free energy, but also indicates that other sequence effects may play a role. All base pairs are observed to have a propensity for opening toward the major groove. The preferred opening base is cytosine for GC base pairs, while for AT there is sequence dependent competition between the two bases. For AT opening, we identify two non-canonical base pair interactions contributing to a local minimum in the free energy profile. For both AT and CG we observe long-lived interactions with water and with sodium ions at specific sites on the open base pair.

The free energy of locking a ring: Changing a deoxyribonucleoside to a locked nucleic acid

Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3‐step (easy to implement) and a 1‐step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy. We validate the approach on the solvation free energy difference for the nucleosides thymidine, cytosine, and 5‐methyl‐cytosine.

Effect of mutations on internal dynamics of an RNA hairpin from hepatitis B virus

Conformational dynamics plays a key role in mediating specific interactions between RNAs and proteins. Flexible parts, such as the loop regions, are often involved in protein binding. Characterization of the factors that influence the flexibility of loop regions will improve our understanding of RNA-protein binding. Here we use molecular dynamics simulations to study the dynamical features of the apical stem-loop of hepatitis B virus and a mutant, with two consensus-based secondary structure mutations (A-U→C-G) in the stem region. The mutations reduce the dynamics of the system and influence the hairpin conformations. The simulations show that inducing rigidity in the stem affects the loop conformational flexibility: the loop residues become less mobile and less accessible to the solvent, and thus less accessible to a possible targeting protein.