Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Molecular Dynamics Simulations Elucidate the Molecular Basis of Pre-mRNA Translocation by the Prp2 Spliceosomal Helicase.

The spliceosome machinery catalyzes precursor-messenger RNA (pre-mRNA) splicing by undergoing at each splicing cycle assembly, activation, catalysis, and disassembly processes, thanks to the concerted action of specific RNA-dependent ATPases/helicases. Prp2, a member of the DExH-box ATPase/helicase family, harnesses the energy of ATP hydrolysis to translocate a single pre-mRNA strand in the 5' to 3' direction, thus promoting spliceosome remodeling to its catalytic-competent state. Here, we established the functional coupling between ATPase and helicase activities of Prp2. Namely, extensive multi-µs molecular dynamics simulations allowed us to unlock how, after pre-mRNA selection, ATP binding, hydrolysis, and dissociation induce a functional typewriter-like rotation of the Prp2 C-terminal domain. This movement, endorsed by an iterative swing of interactions established between specific Prp2 residues with the nucleobases at 5'- and 3'-ends of pre-mRNA, promotes pre-mRNA translocation. Notably, some of these Prp2 residues are conserved in the DExH-box family, suggesting that the translocation mechanism elucidated here may be applicable to all DExH-box helicases.

Molecular Basis of RNA-Driven ATP Hydrolysis in DExH-Box Helicases.

The spliceosome machinery catalyzes precursor messenger (pre-m)RNA splicing. In each cycle, the spliceosome experiences massive compositional and conformational remodeling fueled by the concerted action of specific RNA-dependent ATPases/helicases. Intriguingly, these enzymes are allosterically activated to perform ATP hydrolysis and trigger helicase activity only upon pre-mRNA binding. Yet, the molecular mechanism underlying the RNA-driven regulation of their ATPase function remains elusive. Here, we focus on the Prp2 ATPase/helicase which contributes to reshaping the spliceosome into its catalytic competent state. By performing classical and quantum-classical molecular dynamics simulations, we unprecedentedly unlock the molecular terms governing the Prp2 ATPase/helicase function. Namely, we dissect the molecular mechanism of ATP hydrolysis, and we disclose that RNA binding allosterically triggers the formation of a set of interactions linking the RNA binding tunnel to the catalytic site. This activates the Prp2's ATPase function by optimally placing the nucleophilic water and the general base of the enzymatic process to perform ATP hydrolysis. The key structural motifs, mechanically coupling RNA gripping and the ATPase/helicase functions, are conserved across all DExH-box helicases. This mechanism could thus be broadly applicable to all DExH-box helicase family.

Computational Study of the Inhibition of RgpB Gingipain, a Promising Target for the Treatment of Alzheimer's Disease.

Alzheimer's disease represents one of the most ambitious challenges for biomedical sciences due to the growing number of cases worldwide in the elderly population and the lack of efficient treatments. One of the recent attempts to develop a treatment points to the cysteine protease RgpB as a promising drug target. In this attempt, several small-molecule covalent inhibitors of this enzyme have been proposed. Here, we report a computational study at the atomic level of the inhibition mechanism of the most promising reported compounds. Molecular dynamics simulations were performed on six of them, and their binding energies in the active site of the protein were computed. Contact maps and interaction energies were decomposed by residues to disclose those key interactions with the enzyme. Finally, quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations were performed to evaluate the reaction mechanism by which these drug candidates lead to covalently bound complexes, inhibiting the RgpB protease. The results provide a guide for future re-design of prospective and efficient inhibitors for the treatment of Alzheimer's disease.

Unrevealing the Proteolytic Activity of RgpB Gingipain from Computational Simulations.

Alzheimer's disease represents one of the greatest medical concerns for today's population and health services. Its multifactorial inherent nature represents a challenge for its treatment and requires the development of a broad spectrum of drugs. Recently, the cysteine protease gingipain RgpB has been related to neurodegenerative diseases, including Alzheimer's disease, and its inhibition appears to be a promising neuroprotective strategy. Given these features, a computational study that integrates molecular dynamics (MD) simulations with classical and hybrid quantum mechanics/molecular mechanics (QM/MM) potentials was carried out to unravel the atomistic details of RgpB activity. First, a preliminary study based on principal component analysis (PCA), determined the protonation state of the Cys/His catalytic dyad, as well as the crucial role of a flexible loop that favors reactive interactions of the catalytic residues and the peptide in the precatalytic state in its closed conformation. Then, different mechanisms were explored by means of QM/MM MD simulations. The most favorable mechanism consists of two stages. First is an acylation stage that takes place in two steps where, initially, the sulfur atom of the C244 residue attacks the carbonylic carbon of the peptide and the proton of the C244 residue is transferred to the amino group of the peptide in a concerted manner. Subsequently, the peptide bond is broken, and a fragment of the peptide is released. After that, the deacylation stage takes place in a single step where a water molecule attacks the carbonylic carbon of the peptide and a proton of the water is transferred to the C244 residue. The free energy barrier of the rate limiting step is in very good agreement with available experimental data. The mechanism exhibits an unusual role of H211 residue compared with other cysteine proteases but a crucial role of the peptide in triggering the catalysis. Notably, the atomic and energetic particularities found represent a significant contribution to the comprehension of the reaction mechanism and a great opportunity for the design of efficient inhibitors of gingipain RgpB.

Polymers of ε-Caprolactone Using New Copper(II) and Zinc(II) Complexes as Initiators: Synthesis, Characterization and X-Ray Crystal Structures.

Five of six new Zn(II) and Cu(II) complexes were active in the ring-opening polymerization (ROP) of ε-caprolactone (CL) under solvent-free conditions, producing polycaprolactones (PCLs) of high crystallinity with molecular weights between 22,900 and 38,700 g mol-1 and decomposition temperatures above 260 °C. ¹H NMR analysis demonstrated that the PCLs obtained were mainly linear, having hydroxymethylene groups at the chain ends. The results obtained indicated a significant improvement in terms of the ratio of monomer:initiator compared to related Cu(II) and Zn(II) complexes. In addition, the structures of the complexes 1 and 4 were determined by single-crystal X-ray diffraction. The synthesis and full characterization of all complexes are described in this paper.