Paolo Ruggerone

First-principles prediction of structure, energetics, formation enthalpy, elastic constants, polarization, and piezoelectric constants of AlN, GaN, and InN: Comparison of local and gradient-corrected density-functional theory

A number of diverse bulk properties of the zinc-blende and wurtzite III-V nitrides AlN, GaN, and InN, are predicted from first principles within density-functional theory using the plane-wave ultrasoft pseudopotential method, within both the local density approximation (LDA) and generalized gradient approximation (GGA) to the exchange-correlation functional. Besides structure and cohesion, we study formation enthalpies (a key ingredient in predicting defect solubilities and surface stability), spontaneous polarizations and piezoelectric constants (central parameters for nanostructure modeling), and elastic constants. Our study bears out the relative merits of the two density-functional approaches in describing diverse properties of the III-V nitrides (and of the parent species

Functional Rotation of the Transporter AcrB: Insights into Drug Extrusion from Simulations

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AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

Al, Ga, and In). None of the two schemes gives entirely successful results. However, the GGA associated with the multiprojector ultrasoft pseudopotential method slightly outperforms the LDA overall as to lattice parameters, cohesive energies, and formation enthalpies of wurtzite nitrides. This is relevant to the study of properties such as polarization, vibrational frequencies, elastic constants, nonstochiometric substitution, and absorption. A major exception is the formation enthalpy of InN, which is underestimated by the GGA (\ensuremath{\sim}0 vs -0.2 eV).

RND efflux pumps: structural information translated into function and inhibition mechanisms.

The tripartite complex AcrAB-TolC is the major efflux system in Escherichia coli. It extrudes a wide spectrum of noxious compounds out of the bacterium, including many antibiotics. Its active part, the homotrimeric transporter AcrB, is responsible for the selective binding of substrates and energy transduction. Based on available crystal structures and biochemical data, the transport of substrates by AcrB has been proposed to take place via a functional rotation, in which each monomer assumes a particular conformation. However, there is no molecular-level description of the conformational changes associated with the rotation and their connection to drug extrusion. To obtain insights thereon, we have performed extensive targeted molecular dynamics simulations mimicking the functional rotation of AcrB containing doxorubicin, one of the two substrates that were co-crystallized so far. The simulations, including almost half a million atoms, have been used to test several hypotheses concerning the structure-dynamics-function relationship of this transporter. Our results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. Furthermore, we provide strong evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. A concerted opening of the channel between the binding pocket and the gate further favors the displacement of the drug. This microscopically well-funded information allows one to identify the role of specific amino acids during the transitions and to shed light on the functioning of AcrB.

Effect of the F610A Mutation on Substrate Extrusion in the AcrB Transporter: Explanation and Rationale by Molecular Dynamics Simulations

Significance Genome sequencing of a multidrug-resistant clinical isolate of Salmonella Typhimurium from a patient that failed ciprofloxacin therapy revealed a mutation in the efflux pump gene, acrB. Computational modelling revealed that the G288D substitution changed the binding of drugs to the distal binding pocket of AcrB. The mutation was recreated in an unrelated Salmonella strain and also in Escherichia coli; in both species the efflux of ciprofloxacin was increased by the mutation, explaining its resistant phenotype. This is the first time a substitution within an efflux pump protein has been shown to cause drug resistance. Importantly, the finding that one amino acid change can cause resistance to some drugs, but susceptibility to others, informs those developing new antibiotics. The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

Copper−1,10-Phenanthroline Complexes Binding to DNA: Structural Predictions from Molecular Simulations

Efflux pumps of the Resistance Nodulation Division (RND) superfamily play a major role in the intrinsic and acquired resistance of Gram-negative pathogens to antibiotics. Moreover, they are largely responsible for multi-drug resistance (MDR) phenomena in these bacteria. The last decade has seen a sharp increase in the number of experimental and computational studies aimed at understanding their functional mechanisms. Most of these studies focused on the RND drug/proton antiporter AcrB, part of the AcrAB-TolC efflux pump actively recognizing and expelling noxious agents from the interior of bacteria. These studies have been focused on the dynamical interactions between AcrB and its substrates and inhibitors, on the details of the proton translocation mechanisms, and on the way AcrB assembles with protein partners to build up a functional pump. In this review we summarize these advances focusing on the role of AcrB.

Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.

CO escape from myoglobin with metadynamics simulations

Copper-1,10-phenanthroline (phen) complexes Cu(phen)(2)(), Cu(2-Clip-phen), and Cu(3-Clip-phen) (Clip = a serinol bridge between the phen parts) are typically employed as DNA-cleaving agents and are now becoming increasingly important for building multifunctional drugs with improved cytotoxic properties. For instance, Cu(3-Clip-phen) has been combined with distamycin-like minor-groove binders and cisplatin-derivatives, leading to promising results. Density Functional Theory (DFT) and docking calculations as well as molecular dynamics (MD) simulations were performed to describe the mode of binding to DNA of these complexes. Our data suggest the minor-groove binding to be more probable than (partial) intercalation and major-groove binding. In addition, it was found that a combination of factors including planarity, van der Waals interactions with DNA, and structural complementarities may be the key for the cleavage efficiency of these copper complexes.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives

ABSTRACT Efflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus, in silico structure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps of Enterobacteriaceae species, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

The Hydrolysis Mechanism of the Anticancer Ruthenium Drugs NAMI-A and ICR Investigated by DFT−PCM Calculations

The relatively small size of myoglobin makes it suitable for the investigation of the ligand escape process in respiratory proteins and, in general, an ideal model system for the study of the more general structure‐function paradigm. In this work, we use Molecular Dynamics simulations combined with an accelerated algorithm, the metadynamics, to probe the escape of CO from myoglobin. Our approach permits to quantitatively describe the escape process via the reconstruction of the associated free energy surface. Additionally, hints on the involvement of a larger numbers of residues than hitherto assumed in the gating process are extracted from our data. Proteins 2008.