MD and QM/MM Study of the Quaternary HutP Homohexamer Complex with mRNA, l‑Histidine Ligand, and Mg²⁺

The HutP protein from <i>B. subtilis</i> regulates histidine metabolism by interacting with an antiterminator mRNA hairpin in response to the binding of l-histidine and Mg²⁺. We studied the functional ligand-bound HutP hexamer complexed with two mRNAs using all-atom microsecond-scale explicit-solvent MD simulations performed with the Amber force fields. The experimentally observed protein-RNA interface exhibited good structural stability in the simulations with the exception of some fluctuations in an unusual adenine-threonine interaction involving two closely spaced H-bonds. We further investigated this interaction by comparing QM/MM and MM optimizations, using the QM region comprising almost 350 atoms described at the DFT-D3 level. The QM/MM method clearly improved the adenine-threonine interaction compared to MM, especially when the X–H bond lengths were frozen during the MM optimization to mimic the use of SHAKE in the MD simulations. Thus, both the MM approximation and the use of SHAKE can compromise the description of H-bonds at protein-RNA interfaces. The simulations also revealed a notable Mg²⁺-parameter dependence in the behavior of the ligand-binding pocket (LBP). With the SPC/E water model, the 12–6 Åqvist and Li&Merz parameters provided an entirely stable LBP structure, but the 12–6 Allnér and 12–6–4 Li&Merz parametrizations resulted in a progressive loss of direct nitrogen–Mg²⁺ LBP coordination. The Allnér and Li&Merz 12–6 parametrizations were also tested with the TIP3P water model; the LBP was destabilized in both cases. This illustrates the difficulty of consistently describing different Mg²⁺ interactions using nonpolarizable force fields. Overall, the simulations support the hypothesis that HutP protein becomes fully structured upon ligand binding. Subsequent RNA binding does not affect the protein structure, in keeping with the mechanism inferred from experimental structures

MD and QM/MM Study of the Quaternary HutP Homohexamer Complex with mRNA, l‑Histidine Ligand, and Mg²⁺

The HutP protein from <i>B. subtilis</i> regulates histidine metabolism by interacting with an antiterminator mRNA hairpin in response to the binding of l-histidine and Mg²⁺. We studied the functional ligand-bound HutP hexamer complexed with two mRNAs using all-atom microsecond-scale explicit-solvent MD simulations performed with the Amber force fields. The experimentally observed protein-RNA interface exhibited good structural stability in the simulations with the exception of some fluctuations in an unusual adenine-threonine interaction involving two closely spaced H-bonds. We further investigated this interaction by comparing QM/MM and MM optimizations, using the QM region comprising almost 350 atoms described at the DFT-D3 level. The QM/MM method clearly improved the adenine-threonine interaction compared to MM, especially when the X–H bond lengths were frozen during the MM optimization to mimic the use of SHAKE in the MD simulations. Thus, both the MM approximation and the use of SHAKE can compromise the description of H-bonds at protein-RNA interfaces. The simulations also revealed a notable Mg²⁺-parameter dependence in the behavior of the ligand-binding pocket (LBP). With the SPC/E water model, the 12–6 Åqvist and Li&Merz parameters provided an entirely stable LBP structure, but the 12–6 Allnér and 12–6–4 Li&Merz parametrizations resulted in a progressive loss of direct nitrogen–Mg²⁺ LBP coordination. The Allnér and Li&Merz 12–6 parametrizations were also tested with the TIP3P water model; the LBP was destabilized in both cases. This illustrates the difficulty of consistently describing different Mg²⁺ interactions using nonpolarizable force fields. Overall, the simulations support the hypothesis that HutP protein becomes fully structured upon ligand binding. Subsequent RNA binding does not affect the protein structure, in keeping with the mechanism inferred from experimental structures

Bioinformatics and Molecular Dynamics Simulation Study of L1 Stalk Non-Canonical rRNA Elements: Kink-Turns, Loops, and Tetraloops

The L1 stalk is a prominent mobile element of the large ribosomal subunit. We explore the structure and dynamics of its non-canonical rRNA elements, which include two kink-turns, an internal loop, and a tetraloop. We use bioinformatics to identify the L1 stalk RNA conservation patterns and carry out over 11.5 μs of MD simulations for a set of systems ranging from isolated RNA building blocks up to complexes of L1 stalk rRNA with the L1 protein and tRNA fragment. We show that the L1 stalk tetraloop has an unusual GNNA or UNNG conservation pattern deviating from major GNRA and YNMG RNA tetraloop families. We suggest that this deviation is related to a highly conserved tertiary contact within the L1 stalk. The available X-ray structures contain only UCCG tetraloops which in addition differ in orientation (<i>anti</i> vs <i>syn</i>) of the guanine. Our analysis suggests that the <i>anti</i> orientation might be a mis-refinement, although even the <i>anti</i> interaction would be compatible with the sequence pattern and observed tertiary interaction. Alternatively, the <i>anti</i> conformation may be a real substate whose population could be pH-dependent, since the guanine <i>syn</i> orientation requires protonation of cytosine in the tertiary contact. In absence of structural data, we use molecular modeling to explore the GCCA tetraloop that is dominant in bacteria and suggest that the GCCA tetraloop is structurally similar to the YNMG tetraloop. Kink-turn Kt-77 is unusual due to its 11-nucleotide bulge. The simulations indicate that the long bulge is a stalk-specific eight-nucleotide insertion into consensual kink-turn only subtly modifying its structural dynamics. We discuss a possible evolutionary role of helix H78 and a mechanism of L1 stalk interaction with tRNA. We also assess the simulation methodology. The simulations provide a good description of the studied systems with the latest bsc0χ_OL3 force field showing improved performance. Still, even bsc0χ_OL3 is unable to fully stabilize an essential sugar-edge H-bond between the bulge and non-canonical stem of the kink-turn. Inclusion of Mg²⁺ ions may deteriorate the simulations. On the other hand, monovalent ions can in simulations readily occupy experimental Mg²⁺ binding sites

Effect of Guanine to Inosine Substitution on Stability of Canonical DNA and RNA Duplexes: Molecular Dynamics Thermodynamics Integration Study

Guanine to inosine (G → I) substitution has often been used to study various properties of nucleic acids. Inosine differs from guanine only by loss of the N2 amino group, while both bases have similar electrostatic potentials. Therefore, G → I substitution appears to be optimally suited to probe structural and thermodynamics effects of single H-bonds and atomic groups. However, recent experiments have revealed substantial difference in free energy impact of G → I substitution in the context of B-DNA and A-RNA canonical helices, suggesting that the free energy changes reflect context-dependent balance of energy contributions rather than intrinsic strength of a single H-bond. In the present study, we complement the experiments by free energy computations using thermodynamics integration method based on extended explicit solvent molecular dynamics simulations. The computations successfully reproduce the basic qualitative difference in free energy impact of G → I substitution in B-DNA and A-RNA helices although the magnitude of the effect is somewhat underestimated. The computations, however, do not reproduce the salt dependence of the free energy changes. We tentatively suggest that the different effect of G → I substitution in A-RNA and B-DNA may be related to different topologies of these helices, which affect the electrostatic interactions between the base pairs and the negatively charged backbone. Limitations of the computations are briefly discussed

Microsecond-Scale MD Simulations of HIV‑1 DIS Kissing-Loop Complexes Predict Bulged-In Conformation of the Bulged Bases and Reveal Interesting Differences between Available Variants of the AMBER RNA Force Fields

We report an extensive set of explicit solvent molecular dynamics (MD) simulations (∼25 μs of accumulated simulation time) of the RNA kissing-loop complex of the HIV-1 virus initiation dimerization site. Despite many structural investigations by X-ray, NMR, and MD techniques, the position of the bulged purines of the kissing complex has not been unambiguously resolved. The X-ray structures consistently show bulged-out positions of the unpaired bases, while several NMR studies show bulged-in conformations. The NMR studies are, however, mutually inconsistent regarding the exact orientations of the bases. The earlier simulation studies predicted the bulged-out conformation; however, this finding could have been biased by the short simulation time scales. Our microsecond-long simulations reveal that all unpaired bases of the kissing-loop complex stay preferably in the interior of the kissing-loop complex. The MD results are discussed in the context of the available experimental data and we suggest that both conformations are biochemically relevant. We also show that MD provides a quite satisfactory description of this RNA system, contrasting recent reports of unsatisfactory performance of the RNA force fields for smaller systems such as tetranucleotides and tetraloops. We explain this by the fact that the kissing complex is primarily stabilized by an extensive network of Watson–Crick interactions which are rather well described by the force fields. We tested several different sets of water/ion parameters but they all lead to consistent results. However, we demonstrate that a recently suggested modification of van der Waals interactions of the Cornell et al. force field deteriorates the description of the kissing complex by the loss of key stacking interactions stabilizing the interhelical junction and excessive hydrogen-bonding interactions

Microsecond-Scale MD Simulations of HIV‑1 DIS Kissing-Loop Complexes Predict Bulged-In Conformation of the Bulged Bases and Reveal Interesting Differences between Available Variants of the AMBER RNA Force Fields

We report an extensive set of explicit solvent molecular dynamics (MD) simulations (∼25 μs of accumulated simulation time) of the RNA kissing-loop complex of the HIV-1 virus initiation dimerization site. Despite many structural investigations by X-ray, NMR, and MD techniques, the position of the bulged purines of the kissing complex has not been unambiguously resolved. The X-ray structures consistently show bulged-out positions of the unpaired bases, while several NMR studies show bulged-in conformations. The NMR studies are, however, mutually inconsistent regarding the exact orientations of the bases. The earlier simulation studies predicted the bulged-out conformation; however, this finding could have been biased by the short simulation time scales. Our microsecond-long simulations reveal that all unpaired bases of the kissing-loop complex stay preferably in the interior of the kissing-loop complex. The MD results are discussed in the context of the available experimental data and we suggest that both conformations are biochemically relevant. We also show that MD provides a quite satisfactory description of this RNA system, contrasting recent reports of unsatisfactory performance of the RNA force fields for smaller systems such as tetranucleotides and tetraloops. We explain this by the fact that the kissing complex is primarily stabilized by an extensive network of Watson–Crick interactions which are rather well described by the force fields. We tested several different sets of water/ion parameters but they all lead to consistent results. However, we demonstrate that a recently suggested modification of van der Waals interactions of the Cornell et al. force field deteriorates the description of the kissing complex by the loss of key stacking interactions stabilizing the interhelical junction and excessive hydrogen-bonding interactions

Atomistic Picture of Opening–Closing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations

Holliday junction (HJ) is a noncanonical four-way DNA structure with a prominent role in DNA repair, recombination, and DNA nanotechnology. By rearranging its four arms, HJ can adopt either closed or open state. With enzymes typically recognizing only a single state, acquiring detailed knowledge of the rearrangement process is an important step toward fully understanding the biological function of HJs. Here, we carried out standard all-atom molecular dynamics (MD) simulations of the spontaneous opening–closing transitions, which revealed complex conformational transitions of HJs with an involvement of previously unconsidered “half-closed” intermediates. Detailed free-energy landscapes of the transitions were obtained by sophisticated enhanced sampling simulations. Because the force field overstabilizes the closed conformation of HJs, we developed a system-specific modification which for the first time allows the observation of spontaneous opening–closing HJ transitions in unbiased MD simulations and opens the possibilities for more accurate HJ computational studies of biological processes and nanomaterials

Atomistic Picture of Opening–Closing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations

Holliday junction (HJ) is a noncanonical four-way DNA structure with a prominent role in DNA repair, recombination, and DNA nanotechnology. By rearranging its four arms, HJ can adopt either closed or open state. With enzymes typically recognizing only a single state, acquiring detailed knowledge of the rearrangement process is an important step toward fully understanding the biological function of HJs. Here, we carried out standard all-atom molecular dynamics (MD) simulations of the spontaneous opening–closing transitions, which revealed complex conformational transitions of HJs with an involvement of previously unconsidered “half-closed” intermediates. Detailed free-energy landscapes of the transitions were obtained by sophisticated enhanced sampling simulations. Because the force field overstabilizes the closed conformation of HJs, we developed a system-specific modification which for the first time allows the observation of spontaneous opening–closing HJ transitions in unbiased MD simulations and opens the possibilities for more accurate HJ computational studies of biological processes and nanomaterials

Reference Simulations of Noncanonical Nucleic Acids with Different χ Variants of the AMBER Force Field: Quadruplex DNA, Quadruplex RNA, and Z-DNA

Refinement of empirical force fields for nucleic acids requires their extensive testing using as wide range of systems as possible. However, finding unambiguous reference data is not easy. In this paper, we analyze four systems that we suggest should be included in standard portfolio of molecules to test nucleic acids force fields, namely, parallel and antiparallel stranded DNA guanine quadruplex stems, RNA quadruplex stem, and Z-DNA. We highlight parameters that should be monitored to assess the force field performance. The work is primarily based on 8.4 μs of 100–250 ns trajectories analyzed in detail followed by 9.6 μs of additional selected backup trajectories that were monitored to verify that the results of the initial analyses are correct. Four versions of the Cornell et al. AMBER force field are tested, including an entirely new parmχ_OL4 variant with χ dihedral specifically reparametrized for DNA molecules containing <i>syn</i>-nucleotides. We test also different water models and ion conditions. While improvement for DNA quadruplexes is visible, the force fields still do not fully reproduce the intricate Z-DNA backbone conformation

Reference Simulations of Noncanonical Nucleic Acids with Different χ Variants of the AMBER Force Field: Quadruplex DNA, Quadruplex RNA, and Z-DNA

Refinement of empirical force fields for nucleic acids requires their extensive testing using as wide range of systems as possible. However, finding unambiguous reference data is not easy. In this paper, we analyze four systems that we suggest should be included in standard portfolio of molecules to test nucleic acids force fields, namely, parallel and antiparallel stranded DNA guanine quadruplex stems, RNA quadruplex stem, and Z-DNA. We highlight parameters that should be monitored to assess the force field performance. The work is primarily based on 8.4 μs of 100–250 ns trajectories analyzed in detail followed by 9.6 μs of additional selected backup trajectories that were monitored to verify that the results of the initial analyses are correct. Four versions of the Cornell et al. AMBER force field are tested, including an entirely new parmχ_OL4 variant with χ dihedral specifically reparametrized for DNA molecules containing <i>syn</i>-nucleotides. We test also different water models and ion conditions. While improvement for DNA quadruplexes is visible, the force fields still do not fully reproduce the intricate Z-DNA backbone conformation