Thereza A. Soares

Validation of the 53A6 GROMOS force field

The quality of biomolecular dynamics simulations relies critically on the force field that is used to describe the interactions between particles in the system. Force fields, which are generally parameterized using experimental data on small molecules, can only prove themselves in realistic simulations of relevant biomolecular systems. In this work, we begin the validation of the new 53A6 GROMOS parameter set by examining three test cases. Simulations of the well-studied 129 residue protein hen egg-white lysozyme, of the DNA dodecamer d(CGCGAATTCGCG)2, and a proteinogenic β3-dodecapeptide were performed and analysed. It was found that the new parameter set performs as well as the previous parameter sets in terms of protein (45A3) and DNA (45A4) stability and that it is better at describing the folding–unfolding balance of the peptide. The latter is a property that is directly associated with the free enthalpy of hydration, to which the 53A6 parameter set was parameterized.

An improved nucleic acid parameter set for the GROMOS force field

Over the past decades, the GROMOS force field for biomolecular simulation has primarily been developed for performing molecular dynamics (MD) simulations of polypeptides and, to a lesser extent, sugars. When applied to DNA, the 43A1 and 45A3 parameter sets of the years 1996 and 2001 produced rather flexible double‐helical structures, in which the Watson–Crick hydrogen‐bonding content was more limited than expected. To improve on the currently available parameter sets, the nucleotide backbone torsional‐angle parameters and the charge distribution of the nucleotide bases are reconsidered based on quantum‐chemical data. The new 45A4 parameter set resulting from this refinement appears to perform well in terms of reproducing solution NMR data and canonical hydrogen bonding. The deviation between simulated and experimental observables is now of the same order of magnitude as the uncertainty in the experimental values themselves.

Cytotoxicity and slow release of the anti-cancer drug doxorubicin from ZIF-8

Metal–organic frameworks are emerging as a powerful platform for the delivery and controlled release of several drug molecules. Herein, we report the incorporation of the anti-cancer drug doxorubicin into the zeolitic imidazolate framework (ZIF-8) with high-load and progressive release. Adsorption measurements show that doxorubicin is incorporated into ZIF-8 with a load of 0.049 g doxorubicin g−1 dehydrated ZIF-8. Doxorubicin is released in a highly controlled and progressive fashion with 66% of the drug released after 30 days. We also characterize the antitumoral potential and cytotoxicity of the doxorubicin-ZIF-8 (DOXO-ZIF-8) complex towards the mucoepidermoid carcinoma of human lung (NCI-H292), human colorectal adenocarcinoma (HT-29), and human promyelocytic leukemia (HL-60) cell lines. It is shown that the complex doxorubicin-ZIF-8 exhibits lower cytotoxicity than pure doxorubicin for the tested cells, possibly due to the slower release of the incorporated drug. Furthermore, host–guest interactions have been addressed from a microscopic perspective through molecular docking simulations. In conjunction with our experimental characterization, the calculations suggest that doxorubicin binds preferentially to the surface rather than into the pores of ZIF-8, whose entry diameter is at least half the size of the shortest axis of the drug. These findings are also consistent with high-resolution X-ray crystallography and NMR spectroscopy studies of ZIF-8 which shows that this framework is very rigid under constant pressure in contrast to previous experimental and theoretical studies of ZIF-8 under gas pressure.

A Glycam-Based Force Field for Simulations of Lipopolysaccharide Membranes: Parametrization and Validation

Lipopolysaccharides (LPS) comprise the outermost layer of the Gram-negative bacteria cell envelope. Packed onto a lipid layer, the outer membrane displays remarkable physical-chemical differences compared to cell membranes. The carbohydrate-rich region confers a membrane asymmetry that underlies many biological processes such as endotoxicity, antibiotic resistance, and cell adhesion. Furthermore, unlike membrane proteins from other sources, integral outer-membrane proteins do not consist of transmembrane α helices; instead they consist of antiparallel β-barrels, which highlights the importance of the LPS membrane as a medium. In this work, we present an extension of the GLYCAM06 force field that has been specifically developed for LPS membranes using our Wolf2Pack program. This new set of parameters for lipopolysaccharide molecules expands the GLYCAM06 repertoire of monosaccharides to include phosphorylated N- and O-acetylglucosamine, 3-deoxy-d-manno-oct-2-ulosonic acid, l-glycero-D-manno-heptose and its O-carbamoylated variant, and N-alanine-d-galactosamine. A total of 1 μs of molecular dynamics simulations of the rough LPS membrane of Pseudomonas aeruginosa PA01 is used to showcase the added parameter set. The equilibration of the LPS membrane is shown to be significantly slower compared to phospholipid membranes, on the order of 500 ns. It is further shown that water molecules penetrate the hydrocarbon region up to the terminal methyl groups, much deeper than commonly observed for phospholipid bilayers, and in agreement with neutron diffraction measurements. A comparison of simulated structural, dynamical, and electrostatic properties against corresponding experimentally available data shows that the present parameter set reproduces well the overall structure and the permeability of LPS membranes in the liquid-crystalline phase.

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation.

The N‐terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight‐stranded antiparallel β‐barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side‐chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel. Proteins 2009.

Investigations on human immunodeficiency virus type 1 integrase/DNA binding interactions via molecular dynamics and electrostatics calculations

The complete three-dimensional structure of the active site region of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) is not unambiguously known. This region includes a flexible loop comprising residues 141-148 and the N-terminal portion of the helix alpha-4, which contains E152, the third catalytic residue, and Y143, which plays a secondary role in catalysis. Relatively high B-factors exist for most of the residues in the aforementioned region. The HIV-1 IN belongs to the polynucleotidyl transferase superfamily, whose members have been proposed to use two divalent metal ions for catalysis. Although only the position of the first metal ion has been determined crystallographically for the HIV-1 IN, we recently have proposed a binding site for the second metal ion. Based on this information, we have performed two 500-psec molecular dynamics simulations of the catalytic domain of the HIV-1 IN containing two Mg(2)+ ions. In one of the simulations, we included a dianionic phosphate group (HPO(4)(2)-) in the active site to mimic a portion of the DNA backbone of a substrate for the integration reaction. Electrostatics calculations and ionization state predictions were carried out on representative structures taken from the molecular dynamics simulations. Different conformational behaviors of the enzyme were observed, depending upon whether two Mg(2)+ ions were bound or two Mg(2)+ ions plus phosphate. The electrostatic calculations performed on the dynamical structures provide a further refinement about which regions of the catalytic domain of the HIV-1 IN may be involved in the DNA binding.

The Role of Nonbonded Interactions in the Conformational Dynamics of Organophosphorous Hydrolase Adsorbed onto Functionalized Mesoporous Silica Surfaces

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Assessment of the convergence of molecular dynamics simulations of lipopolysaccharide membranes

The outer membrane of Gram-negative bacteria is composed of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The chemical structure of LPS results in an asymmetric character of outer membranes that has been shown to play an important role in the electrical properties of porins, low permeability and intrinsic antibiotic resistance of Gram-negative bacteria. Atomistic molecular dynamics simulations of two different configurations of the outer membrane of Pseudomonas aeruginosa under periodic boundary conditions were carried out in order to (1) validate model-derived properties against the available experimental data, (2) identify the properties whose dynamics can be sampled on nanosecond timescales, and (3) evaluate the dependence of the convergence of structural and dynamical properties on the initial configuration of the system, within the chosen force field and simulation conditions. Because the relaxation times associated with the motions of individual LPS monomers in outer membranes are very long, the two initial configurations do not converge to a common ensemble of configuration on the nanosecond time scale. However, a number of properties of the outer membrane that will significantly impact the structural and internal dynamics of transmembrane proteins, most notably the electrostatic potential and molecular density, do converge within the simulated time scale. For these properties, a good agreement with the available experimental data was found. Such a molecular model, capable of accounting for the high asymmetry and low fluidity characteristics of outer membranes provides a more appropriate environment for atomistic simulations of outer membrane proteins.

Electrostatics and flexibility drive membrane recognition and early penetration by the antimicrobial peptide dendrimer bH1

Molecular dynamics simulations of the polycationic antimicrobial peptide dendrimer (Leu)8(DapLeu)4(DapPhe)2DapLys-NH2 binding to membranes suggest that electrostatic interactions with the polyanionic lipopolysaccharide (LPS) and conformational flexibility of the 2,3-diaminopropanoic acid (Dap) branching units drive its selective insertion into microbial membranes.

Ionization state and molecular docking studies for the macrophage migration inhibitory factor: the role of lysine 32 in the catalytic mechanism

The macrophage migration inhibitory factor (MIF) is a cytokine that is structurally similar to certain isomerases and for which multiple immune and catalytic roles have been proposed. Different catalytic activities have been reported for MIF, yet the exact mechanism by which MIF acts is not completely known. As a tautomerase, the enzyme uses a general acid–base mechanism of proton transfer in which the amino‐terminal proline has been shown to function as the catalytic base. We report the results of molecular docking simulations of macrophage migration inhibitory factor with three substrates, D‐dopachrome, L‐dopachrome methyl ester and p‐(hydroxyphenyl)pyruvate. Electrostatic pKa predictions were also performed for the free and complexed forms of the enzyme. The predicted binding mode of p‐(hydroxyphenyl)pyruvate is in agreement with the recently published X‐ray structure. A model for the binding mode of D‐dopachrome and L‐dopachrome methyl ester to MIF is proposed which offers insights into the catalytic mechanism of D‐dopachrome tautomerase activity of MIF. The proposed catalytic mechanism is further supported by the pKa predictions, which suggest that residue Lys32 acts as the general acid for the enzymatic catalysis of D‐dopachrome. Copyright