Noriyuki Kurita

Structure-based analysis of the molecular recognitions between HIV-1 TAR-RNA and transcription factor nuclear factor-kappaB (NFkB).

In this paper we applied the macromolecular docking procedure to perform molecular modeling with the aim of screening transcription factor sequences for possible interaction to the HIV-1 TAR-RNA, employing the software Hex version 4.2. The molecular modeling data were compared with electrophoretic mobility shift assays (EMSA) and surface plasmon resonance (SPR) based biospecific interaction analysis (BIA) using an optical biosensor. Finally the specific interactions between NF-κB and RNA have been calculated utilizing the AMBER-MM and FMO calculations. The results obtained clearly indicate that (a) NF-kB p50 transcription factor can bind TAR-RNA; (b) this binding efficiency is lower than that displayed by NF-kB factor in respect to DNA sequences; (c) other structured RNAs used as controls do not bind to NF-kB; (d) TAR-RNA is capable to bind pre-formed NF-kB/DNA complexes. Despite the fact that our data do not indicate whether NF-kB/TAR-RNA complexes play a role in the early steps of HIV-1 transcriptional activation, the results presented strongly indicate that interactions between transcription factors recruited at the level of HIV-1 LTR might interact with the TAR-RNA and deserve further studies aimed to determine its role in the HIV-1 life cycle.

Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti‐inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti‐inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA.

Attacking mechanism of hydroxyl radical to DNA base-pair: density functional study in vacuum and in water.

Recently, the influence of radiation on human body has been recognized as a serious problem. In particular, highly reactive hydroxyl radicals produced by the radiation react with DNA, resulting in a great damage on its structure and electronic properties. It is thus important to investigate the reaction mechanism of to DNA for elucidating the initial damage in DNA induced by the radiation. In the present study, we search for transition states (TS) of the reaction between G–C/A–T base-pair and in vacuum and in water, by the density functional theory (DFT) calculations. At first, we obtain the stable structures for the dehydrogenated G–C and A–T, in which the hydrogen atom of NH2 group of G or A base is abstracted by . From the structures of the dehydrogenated as well as the natural base-pairs, the TS between these structures is searched for and the activation free energy (AFE) is estimated for the reaction. In vacuum, AFEs for the G–C and A–T are almost the same each other, while the stabilization energy by the reaction for G–C is about 4.9 kcal/mol larger than that for A–T, indicating that the population of the dehydrogenated G–C is remarkably larger than that of the dehydrogenated A–T in vacuum. On the other hand, in water approximated by the continuum solvation model, the AFE for A–T is 2.6 kcal/mol smaller than that for G–C, indicating that the reaction dehydrogenated by occurs more frequently for the solvated A–T base–pair than G–C.

Change in specific interactions between lactose repressor protein and DNA induced by ligand binding: molecular dynamics and molecular orbital calculations

Lactose repressor protein (LacR) plays an essential role in controlling the transcription mechanism of genomic information from DNA to mRNA. It has been elucidated that a ligand binding to LacR regulates allosterically the specific interactions between LacR and operator DNA. However, the effect of the ligand binding on the specific interactions has not been clarified at an atomic level. In this study, we performed classical molecular dynamics (MD) and ab initio fragment molecular orbital simulations to elucidate the effect of ligand binding at atomic and electronic levels. The MD simulations for the solvated complexes with LacR-dimer, DNA and ligand demonstrate that the binding of an inducer IPTG to LacR-dimer significantly changes the structure of LacR-monomer to cause strong interactions between LacR-monomers, resulting in weakening the interactions between LacR-dimer and DNA. In contrast, the binding of an anti-inducer ONPF to LacR-dimer was found to enhance the interactions between LacR-dimer and DNA. These findings are consistent with the functions of IPTG and ONPF as an inducer and an anti-inducer, respectively. We therefore proposed a simplified model for the effect of the ligand binding on the specific interactions between LacR-dimer and DNA.

The effects of vitronectin on specific interactions between urokinase-type plasminogen activator and its receptor: ab initio molecular orbital calculations

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on the surface of a cancer cell is considered to be a trigger for starting cancer invasions. In addition, the somatomedin B (SMB) domain of vitronectin binds simultaneously to uPAR to construct a ternary complex of uPAR–uPA–SMB. Here we present stable structures of the solvated complexes of uPAR–uPA and uPAR–uPA–SMB obtained by classical molecular mechanics simulations, and the specific interactions between uPAR, uPA and SMB are investigated by ab initio fragment molecular orbital calculations. The result indicates that the SMB binding enhances the binding affinity between uPAR and uPA, although there is no direct contact between SMB and uPA. In particular, the specific interaction between uPAR and the Lys36 residue of uPA is significantly affected by the SMB binding. The positively charged Lys23, Lys46 and Lys61 residues of uPA have strong attractive interactions to uPAR in both the uPAR–uPA and uPAR–uPA–SMB complexes, demonstrating the importance of these residues in the specific binding between uPAR and uPA. The current results on the specific interactions are informative for proposing potent antagonists, which block the uPA and SMB bindings to uPAR.

Influence of solvating water molecules on the attacking mechanisms of OH-radical to DNA base pairs: DFT calculations in explicit waters

To elucidate the influence of solvating water molecules on the attacking mechanism of OH-radical to DNA base pairs (G–C and A–T), we investigated the reaction mechanism in water, by the use of the density functional theory (DFT) calculations by considering water molecules explicitly. The results reveal that OH-radical is stabilized near the NH2 group of cytosine of G–C by the water molecules hydrogen bonded to the OH-radical and that 2.5xa0kcal/mol activation free energy is needed for extracting the hydrogen atom from the NH2 group. On the other hand, OH-radical prefers to extract the hydrogen atom from the NH2 group of adenine in the solvated A–T. As for the tautomeric reaction of the base pair attacked by OH-radical, we found the transition state for the reaction from A to T to its tautomeric form A*–T*, although the activation free energy is rather large (25xa0kcal/mol). By contrast, in the G–C attacked by OH-radical, one central proton can move freely from G to C, resulting in the tautomeric form G*–C*. Therefore, our DFT calculations in explicit water molecules elucidate the possibility that the attacking of OH-radical to G–C causes its tautomeric form G*–C*, while A–T attacked by OH-radical cannot transform into its A*–T* form in a normal condition. This finding will be useful for predicting the effect of OH-radical on the genetic information recorded in DNA base sequences.

Ab initio molecular simulations for proposing novel peptide inhibitors blocking the ligand-binding pocket of urokinase receptor

Recent biochemical experiments have revealed that a variety of proteases play important roles in cancer invasion and metastasis. Among these proteases, urokinase-type plasminogen activator (uPA) is particularly important, since its specific binding to the receptor (uPAR) existing on the surface of a cancer cell is considered to be a trigger for cancer invasion. It is thus expected that the blocking of the binding can inhibit cancer invasion in the cancer patients and improve their prognosis dramatically. To develop a potent inhibitor for the binding, many types of peptides of amino acids were produced and their effect on the cancer invasion was investigated in the previous biochemical experiments. On the other hand, our previous ab initio molecular simulations have clarified that some amino acid residues of uPA play important roles in the specific binding between uPA and uPAR. In the present study, we propose some peptides composed of these important residues and investigate the specific interactions and the binding affinity between uPAR and the peptides at an electronic level, using ab initio molecular simulations. Base on the results simulated, we elucidate which peptide can bind more strongly to uPAR and propose a novel potent peptide which can inhibit the binding between uPAR and uPA efficiently.

Effect of BrU on the transition between wobble Gua-Thy and tautomeric Gua-Thy base-pairs: ab initio molecular orbital calculations

We investigated transition states (TS) between wobble Guanine-Thymine (wG-T) and tautomeric G-T base-pair as well as Br-containing base-pairs by MP2 and density functional theory (DFT) calculations. The obtained TS between wG-T and G*-T (asterisk is an enol-form of base) is different from TS got by the previous DFT calculation. The activation energy (17.9 kcal/mol) evaluated by our calculation is significantly smaller than that (39.21 kcal/mol) obtained by the previous calculation, indicating that our TS is more preferable. In contrast, the obtained TS and activation energy between wG-T and G-T* are similar to those obtained by the previous DFT calculation. We furthermore found that the activation energy between wG-BrU and tautomeric G-BrU is smaller than that between wG-T and tautomeric G-T. This result elucidates that the replacement of CH3 group of T by Br increases the probability of the transition reaction producing the enol-form G* and T* bases. Because G* prefers to bind to T rather than to C, and T* to G not A, our calculated results reveal that the spontaneous mutation from C to T or from A to G base is accelerated by the introduction of wG-BrU base-pair.

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.