Detecting DNA Mismatches with Metallo-Insertors: A Molecular Simulation Study

Molecules that selectively recognize DNA mismatches (MMs) play a key role as nucleic acids probes and as chemotherapeutic agents. Metallo-insertors bind to the minor groove (mG) of double strand (ds) DNA, expelling the mismatched base pairs and acting as their pi-stacking replacement. In contrast, metallo-intercalators bind to the major groove (MG) of ds DNA and pi-stack to adjacent base pairs. In this study we focused on structural and energetic properties of Delta-[Rh(bpy)(2)(chrysi)](3+) (1), Delta-[Ru(bpy)(2)(ddpz)](2+) (2), and Delta-[Ru(bpy)(2)(eilatin)](2+) (3) as prototypical examples of metallo-insertors and intercalators. For all molecules we characterized both insertion and intercalation into a DNA dodecamer via force field based molecular dynamics (MD) and hybrid quantum-classical (QM/MM) MD simulations. A structural analysis of the 1-3/DNA noncovalent adducts reveals that the insertion provokes an untwist of the DNA, an opening of the mG and of the phosphate backbone in proximity of the mismatch, while the intercalation induces smaller changes of these structural parameters. This behavior appears to be correlated with the size of the inserting/intercalating ligand in proximity of the metal coordination site. Moreover, our simulations show that the different selectivity of 1 toward distinct MM types may be correlated with the thermodynamic stability of the MMs in the free DNA and with that of the corresponding insertion adduct. Understanding the factors which tune a specific insertion is of crucial importance for designing specific luminescent probes that selectively recognize MMs, as well as for developing more effective anticancer drugs active in MM repair of deficient cells lines

The molecular mechanism of secondary sodium symporters elucidated through the lens of the computational microscope

Transport of molecules across cellular membranes is a key biological process for normal cell function. As such, secondary active transporters exploit electrochemical ion gradients to carry out fundamental processes, i.e. nutrients uptake, ion regulation, neurotransmission, and substrate extrusion. Despite their modest sequence similarity, several Na+ symporters share the same fold of LeuT (leucine transporter), a prokaryotic member of the neurotransmitter-sodium symporter family, pinpointing to a common structural/functional mechanism of transport. This is associated with specific conformational transitions occurring along a so-called alternating access mechanism. Thanks to recent advances in computer simulation techniques and the ever-increasing computational power that has become available in the last decade, molecular dynamics (MD) simulations have been largely employed to provide atomistic insights into mechanistic, kinetic, and thermodynamic aspects of this family of transporters. Here we report a detailed overview of selected Na+-symporters belonging to the LeuT-fold superfamily for which different aspects of the transport mechanism have been addressed using both experimental and computational studies. The aim of this review is to describe current state-of-the-art knowledge on the mechanism of these transporters showing how molecular simulations have contributed to elucidate mechanistic aspects and can provide nowadays a spatial and temporal resolution, allowing the interpretation of experimental findings, complementing biophysical methods, and filling the gaps in fragmentary experimental information

The catalytic mechanism of steroidogenic cytochromes P450 from all-atom simulations: Entwinement with membrane environment, redox partners, and post-transcriptional regulation

Cytochromes P450 (CYP450s) promote the biosynthesis of steroid hormones with major impact on the onset of diseases such as breast and prostate cancers. By merging distinct functions into the same catalytic scaffold, steroidogenic CYP450s enhance complex chemical transformations with extreme efficiency and selectivity. Mammalian CYP450s and their redox partners are membrane-anchored proteins, dynamically associating to form functional machineries. Mounting evidence signifies that environmental factors are strictly intertwined with CYP450s catalysis. Atomic-level simulations have the potential to provide insights into the catalytic mechanism of steroidogenic CYP450s and on its regulation by environmental factors, furnishing information often inaccessible to experimental means. In this review, after an introduction of computational methods commonly employed to tackle these systems, we report the current knowledge on three steroidogenic CYP450s\u2014CYP11A1, CYP17A1, and CYP19A1\u2014endowed with multiple catalytic functions and critically involved in cancer onset. In particular, besides discussing their catalytic mechanisms, we highlight how the membrane environment contributes to (i) regulate ligand channeling through these enzymes, (ii) modulate their interactions with specific protein partners, (iii) mediate post-transcriptional regulation induced by phosphorylation. The results presented set the basis for developing novel therapeutic strategies aimed at fighting diseases originating from steroid metabolism dysfunction

Theoretical Studies of Homogeneous Catalysts Mimicking Nitrogenase

The conversion of molecular nitrogen to ammonia is a key biological and chemical process and represents one of the most challenging topics in chemistry and biology. In Nature the Mo-containing nitrogenase enzymes perform nitrogen 'fixation' via an iron molybdenum cofactor (FeMo-co) under ambient conditions. In contrast, industrially, the Haber-Bosch process reduces molecular nitrogen and hydrogen to ammonia with a heterogeneous iron catalyst under drastic conditions of temperature and pressure. This process accounts for the production of millions of tons of nitrogen compounds used for agricultural and industrial purposes, but the high temperature and pressure required result in a large energy loss, leading to several economic and environmental issues. During the last 40 years many attempts have been made to synthesize simple homogeneous catalysts that can activate dinitrogen under the same mild conditions of the nitrogenase enzymes. Several compounds, almost all containing transition metals, have been shown to bind and activate N(2) to various degrees. However, to date Mo(N(2))(HIPTN)(3)N with (HIPTN)(3)N= hexaisopropyl-terphenyl-triamidoamine is the only compound performing this process catalytically. In this review we describe how Density Functional Theory calculations have been of help in elucidating the reaction mechanisms of the inorganic compounds that activate or fix N(2). These studies provided important insights that rationalize and complement the experimental findings about the reaction mechanisms of known catalysts, predicting the reactivity of new potential catalysts and helping in tailoring new efficient catalytic compounds

Interaction between the DNA model base 9-ethylguanine and a group of ruthenium polypyridyl complexes: Kinetics and conformational temperature dependence

The binding capability of three ruthenium polypyridyl compounds of structural formula [Ru(apy)(tpy)Ln-](ClO4)((2-n)) [1a-c; apy = 2,2'-azobis(pyridine), tpy = 2,2':6',2 ''-terpyridine, L = Cl, H2O, CH3CN] to a fragment of DNA was studied. The interaction between each of these complexes and the DNA model base 9-ethylguanine (9-EtGua) was followed by means of H-1 NMR studies. Density functional theory calculations were carried out to explore the preferential ways of coordination between the ruthenium complexes and guanine. The ruthenium-9-EtGua adduct formed was isolated and fully characterized using different techniques. A variable-temperature H-1 NMR experiment was carried out that showed that while the 9-EtGua fragment was rotating fast at high temperature, a loss of symmetry was suffered by the model base adduct as the temperature was lowered, indicating restricted rotation of the guanine residue

Cancer-Related Mutations Alter RNA-Driven Functional Cross-Talk Underlying Premature-Messenger RNA Recognition by Splicing Factor SF3b

The pillar of faithful premature-messenger (pre-mRNA)splicingis the precise recognition of key intronic sequences by specific splicingfactors. The heptameric splicing factor 3b (SF3b) recognizes the branchpoint sequence (BPS), a key part of the 3 & PRIME; splice site. SF3bcontains SF3B1, a protein holding recurrent cancer-associated mutations.Among these, K700E, the most-frequent SF3B1 mutation, triggers aberrantsplicing, being primarily implicated in hematologic malignancies.Yet, K700E and the BPS recognition site are 60 & ANGS; apart, suggestingthe existence of an allosteric cross-talk between the two distal spots.Here, we couple molecular dynamics simulations and dynamical networktheory analysis to unlock the molecular terms underpinning the impactof SF3b splicing factor mutations on pre-mRNA selection. We establishthat by weakening and remodeling interactions of pre-mRNA with SF3b,K700E scrambles RNA-mediated allosteric cross-talk between the BPSand the mutation site. We propose that the altered allostery contributesto cancer-associated missplicing by mutated SF3B1. This finding broadensour comprehension of the elaborate mechanisms underlying pre-mRNAmetabolism in eukaryotes

Enantioselective palladium-catalyzed hydrosilylation of styrene: Detailed reaction mechanism from first-principles and hybrid QM/MM molecular dynamics simulations

The mechanism of the enantioselective hydrosilylation of styrene catalyzed by Pd-0 species generated in situ from dichloro {1-{(R)-1-[(S)-2(diphenylphosphino-kappaP)ferrocenyl]ethyl}-3-trimethylphenyl-5-1H-pyrazole-kappaN}palladium, 1, has been investigated in detail through ab initio molecular dynamics and hybrid ab initio molecular dynamics/molecular mechanics (QM/MM) calculations. Different QM/MM models have been adopted in order to probe the specific steric and electronic contributions of different substituents. The catalytic cycle is initiated by the formation of a weakly bound pi-complex (DeltaE approximate to -5.4 kcal/mol) under simultaneous detachment of the pyrazole ligand. In agreement with a Chalk-Harrod mechanism, this is followed by the migratory insertion of the hydride, which leads to a eta(3)-coordination mode of the benzylic fragment. The significant stabilization of the allylic intermediate (DeltaE approximate to -11 kcal/mol) is responsible for the high regioselectivity of the reaction (as well as for its enantioselectivity). The rate-determining step with an activation barrier of 16 kcal/mol is the migration of the silyl ligand to the a-carbon of the substrate with concomitant closure of the ligand chelate ring. This step leads to the formation of an intermediate in which the phenyl moiety of the product remains coordinated in an eta(2)-mode to the palladium. The addition of trichlorosilane leads to product formation and hence to the regeneration of the catalyst. A unimolecular reaction pathway on the other hand, in which the transfer of the silyl ligand to the benzylic fragment is concerted with the addition of a molecule of HSiCl3 to the catalyst, is disfavored by an activation barrier of similar to30 kcal/mol

Copper trafficking in eukaryotic systems: current knowledge from experimental and computational efforts

Copper plays a vital role in fundamental cellular functions, and its concentration in the cell must be tightly regulated, as dysfunction of copper homeostasis is linked to severe neurological diseases and cancer. This review provides a compendium of current knowledge regarding the mechanism of copper transfer from the blood system to the Golgi apparatus; this mechanism involves the copper transporter hCtr1, the metallochaperone Atox1, and the ATPases ATP7A/B. We discuss key insights regarding the structural and functional properties of the hCtr1-Atox1-ATP7B cycle, obtained from diverse studies relying on distinct yet complementary biophysical, biochemical, and computational methods. We further address the mechanistic aspects of the cycle that continue to remain elusive. These knowledge gaps must be filled in order to be able to harness our understanding of copper transfer to develop therapeutic approaches with the capacity to modulate copper metabolism

Investigating the molecular mechanism of h3b‐8800: A splicing modulator inducing preferential lethality in spliceosome‐mutant cancers

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre‐mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B‐8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B‐8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome‐mutant cancer cells remains elusive. Here, microsecond‐long all‐atom simulations addressed the binding/dissociation mechanism of H3B‐8800 to wild type and K700E SF3B1‐containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B‐8800 binding. This supports the hypothesis that the selectivity of H3B‐8800 towards mutant cancer cells is unrelated to its preferential targeting ofK700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B‐8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug‐discovery efforts to specifically target mutant SF3b