Multiscale simulations identify origins of differential carbapenem hydrolysis by the OXA-48 β-lactamase

OXA-48 β-lactamases are frequently encountered in bacterial infections caused by carbapenem-resistant Gram-negative bacteria. Due to the importance of carbapenems in treatment of healthcare-associated infections, and the increasingly wide dissemination of OXA-48-like enzymes on plasmids, these β-lactamases are of high clinical significance. Notably, OXA-48 hydrolyses imipenem more efficiently than other commonly used carbapenems, such as meropenem. Here, we use extensive multi-scale simulations of imipenem and meropenem hydrolysis by OXA-48 to dissect the dynamics and to explore differences in reactivity of the possible conformational substates of the respective acylenzymes. QM/MM simulations of the deacylation reaction for both substrates demonstrate that deacylation is favoured when the 6α-hydroxyethyl group is able to hydrogen bond to the water molecule responsible for deacylation, but disfavoured by increasing hydration of either oxygen of the carboxylated Lys73 general base. Differences in free energy barriers calculated from the QM/MM simulations correlate well with the experimentally observed differences in hydrolytic efficiency between meropenem and imipenem. We conclude that the impaired breakdown of meropenem, compared to imipenem, which arises from a subtle change in the hydrogen bonding pattern between the deacylating water molecule and the antibiotic, is most likely induced by the meropenem 1-methyl group. In addition to increased insights into carbapenem breakdown by OXA β-lactamases, which may aid design of new antibiotics or inhibitors, our approach exemplifies the combined use of atomistic simulations in determining the possible different enzyme-substrate substates, and their influence on enzyme reaction kinetics.

Square network based upon the molecular salt of the tetraprotonated photoproduct rtct-tetrakis(pyridin-4-yl)cyclobutane and the sulfate anion

The formation and crystal structure of a hydrated molecular salt that results in a square network is reported. The crystalline solid is based upon the tetraprotonated photoproduct rtct-tetrakis(pyridin-4-yl)cyclobutane (4H- rtct -TPCB)4+ along with two sulfate anions (SO4 2−) and eight waters of hydration, namely, 4,4′,4′′,4′′′-(cyclobutane-1,2,3,4-tetrayl)tetrapyridinium bis(sulfate) octahydrate, C24H24N4 4+·2SO4 2−·8H2O. The fully protonated photoproduct acts as a four-connecting node within the square network by engaging in four charge-assisted N+—H...O hydrogen bonds to the sulfate anion. The observed hydrogen-bonding pattern in this square network is akin to T-silica, which is a metastable form of SiO2. The included water molecules and sulfate anions engage in numerous O—H...O hydrogen bonds to form various hydrogen-bonded ring structures.

Extension of the CL&Pol Polarizable Force Field to Electrolytes, Protic Ionic Liquids and Deep Eutectic Solvents

The polarizable CL&Pol force field presented in our previous study, Transferable, Polarizable Force Field for Ionic Liquids (J. Chem. Theory Comput. 2019, 15, 5858, DOI: 10.1021/acs.jctc.9b00689), is extended to electrolytes, protic ionic liquids, deep eutectic solvents, and glycols. These systems are problematic in polarizable simulations because they contain either small, highly charged ions or strong hydrogen bonds, which cause trajectory instabilities due to the pull exerted on the induced dipoles. We use a Tang-Toennies function to dampen, or smear, the interactions between charges and induced dipole at short range involving small, highly charged atoms (such as hydrogen or lithium), thus preventing the ``polarization catastrophe''. The new force field gives stable trajectories and is validated through comparison with experimental data on density, viscosity, and ion diffusion coefficients of liquid systems of the above-mentioned classes. The results also shed light on the hydrogen-bonding pattern in ethylammonium nitrate, a protic ionic liquid, for which the literature contains conflicting views. We describe the implementation of the Tang-Toennies damping function, of the temperature-grouped Nosé-Hoover thermostat for polarizable molecular dynamics and of the periodic perturbation method for viscosity evaluation from non-equilibrium trajectories in the LAMMPS molecular dynamics code. The main result of this work is the wider applicability of the CL&Pol polarizable force field to new, important classes of fluids, achieving robust trajectories and a good description of equilibrium and transport properties in challenging systems. The fragment-based approach of CL&Pol will allow ready extension to a wide variety of protic ionic liquids, deep eutectic solvents and electrolytes.<br>

The SERCA residue Glu340 mediates interdomain communication that guides Ca2+transport

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA’s 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

Transferable, Polarizable Force Field for Electrolytes, Protic Ionic Liquids and Deep Eutectic Solvents

The transferable, polarizable CL&Pol force field for aprotic ionic liquids presented in our previous study (J. Chem. Theory Comput. 2019, 15, 5858, DOI: 10.1021/acs.jctc.9b00689) is extended to electrolytes, protic ionic liquids, deep eutectic solvents, and glycols. These systems are problematic in polarizable simulations because they contain either small, highly charged ions or strong hydrogen bonds, which cause trajectory instabilities due to the pull exerted on the induced dipoles. We use a Tang-Toennies function to dampen, or smear, the interactions between charges and induced dipole at short range involving small, highly charged atoms (such as hydrogen or lithium), thus preventing the "polarization catastrophe". The new force field gives stable trajectories and is validated through comparison with experimental data on density, viscosity, and ion diffusion coefficients of liquid systems of the above-mentioned classes. The results also shed light on the hydrogen-bonding pattern in ethylammonium nitrate, a protic ionic liquid, for which the literature contains conflicting views. We describe the implementation of the Tang-Toennies damping function, of the temperature-grouped Nosé-Hoover thermostat for polarizable molecular dynamics and of the periodic perturbation method for viscosity evaluation from non-equilibrium trajectories in the LAMMPS molecular dynamics code. The main result of this work is the wider applicability of the CL&Pol polarizable force field to new, important classes of fluids, achieving robust trajectories and a good description of equilibrium and transport properties in challenging systems. The transferability and fragment-based approach of CL&Pol will allow ready extension to a wide variety of protic ionic liquids, deep eutectic solvents and electrolytes.

Transferable, Polarizable Force Field for Electrolytes, Protic Ionic Liquids and Deep Eutectic Solvents

The transferable, polarizable CL&Pol force field for aprotic ionic liquids presented in our previous study (J. Chem. Theory Comput. 2019, 15, 5858, DOI: 10.1021/acs.jctc.9b00689) is extended to electrolytes, protic ionic liquids, deep eutectic solvents, and glycols. These systems are problematic in polarizable simulations because they contain either small, highly charged ions or strong hydrogen bonds, which cause trajectory instabilities due to the pull exerted on the induced dipoles. We use a Tang-Toennies function to dampen, or smear, the interactions between charges and induced dipole at short range involving small, highly charged atoms (such as hydrogen or lithium), thus preventing the "polarization catastrophe". The new force field gives stable trajectories and is validated through comparison with experimental data on density, viscosity, and ion diffusion coefficients of liquid systems of the above-mentioned classes. The results also shed light on the hydrogen-bonding pattern in ethylammonium nitrate, a protic ionic liquid, for which the literature contains conflicting views. We describe the implementation of the Tang-Toennies damping function, of the temperature-grouped Nosé-Hoover thermostat for polarizable molecular dynamics and of the periodic perturbation method for viscosity evaluation from non-equilibrium trajectories in the LAMMPS molecular dynamics code. The main result of this work is the wider applicability of the CL&Pol polarizable force field to new, important classes of fluids, achieving robust trajectories and a good description of equilibrium and transport properties in challenging systems. The transferability and fragment-based approach of CL&Pol will allow ready extension to a wide variety of protic ionic liquids, deep eutectic solvents and electrolytes.

4-(Dimethylamino)benzohydrazide

The title compound, C9H13N3O, crystallizes in the monoclinic space group C2/c and all non-hydrogen atoms are within 0.1 Å of the molecular mean plane. In the crystal, the hydrogen-bonding pattern results in [001] chains built up from fused R 2 2(6) and R 2 2(10) rings; the former consists of N—H...N bonds and the latter N—H...O bonds. Electrostatic and dispersion forces are major contributors to the lattice energy, which was estimated by DFT calculations to be −215.7 kJ mol−1.

Transferable, Polarizable Force Field for Electrolytes, Protic Ionic Liquids and Deep Eutectic Solvents

The transferable, polarizable CL&Pol force field for aprotic ionic liquids presented in our previous study (J. Chem. Theory Comput. 2019, 15, 5858, DOI: 10.1021/acs.jctc.9b00689) is extended to electrolytes, protic ionic liquids, deep eutectic solvents, and glycols. These systems are problematic in polarizable simulations because they contain either small, highly charged ions or strong hydrogen bonds, which cause trajectory instabilities due to the pull exerted on the induced dipoles. We use a Tang-Toennies function to dampen, or smear, the interactions between charges and induced dipole at short range involving small, highly charged atoms (such as hydrogen or lithium), thus preventing the "polarization catastrophe". The new force field gives stable trajectories and is validated through comparison with experimental data on density, viscosity, and ion diffusion coefficients of liquid systems of the above-mentioned classes. The results also shed light on the hydrogen-bonding pattern in ethylammonium nitrate, a protic ionic liquid, for which the literature contains conflicting views. We describe the implementation of the Tang-Toennies damping function, of the temperature-grouped Nosé-Hoover thermostat for polarizable molecular dynamics and of the periodic perturbation method for viscosity evaluation from non-equilibrium trajectories in the LAMMPS molecular dynamics code. The main result of this work is the wider applicability of the CL&Pol polarizable force field to new, important classes of fluids, achieving robust trajectories and a good description of equilibrium and transport properties in challenging systems. The transferability and fragment-based approach of CL&Pol will allow ready extension to a wide variety of protic ionic liquids, deep eutectic solvents and electrolytes.

Transferable, Polarizable Force Field for Electrolytes, Protic Ionic Liquids and Deep Eutectic Solvents

The transferable, polarizable CL&Pol force field for aprotic ionic liquids presented in our previous study (J. Chem. Theory Comput. 2019, 15, 5858, DOI: 10.1021/acs.jctc.9b00689) is extended to electrolytes, protic ionic liquids, deep eutectic solvents, and glycols. These systems are problematic in polarizable simulations because they contain either small, highly charged ions or strong hydrogen bonds, which cause trajectory instabilities due to the pull exerted on the induced dipoles. We use a Tang-Toennies function to dampen, or smear, the interactions between charges and induced dipole at short range involving small, highly charged atoms (such as hydrogen or lithium), thus preventing the "polarization catastrophe". The new force field gives stable trajectories and is validated through comparison with experimental data on density, viscosity, and ion diffusion coefficients of liquid systems of the above-mentioned classes. The results also shed light on the hydrogen-bonding pattern in ethylammonium nitrate, a protic ionic liquid, for which the literature contains conflicting views. We describe the implementation of the Tang-Toennies damping function, of the temperature-grouped Nosé-Hoover thermostat for polarizable molecular dynamics and of the periodic perturbation method for viscosity evaluation from non-equilibrium trajectories in the LAMMPS molecular dynamics code. The main result of this work is the wider applicability of the CL&Pol polarizable force field to new, important classes of fluids, achieving robust trajectories and a good description of equilibrium and transport properties in challenging systems. The transferability and fragment-based approach of CL&Pol will allow ready extension to a wide variety of protic ionic liquids, deep eutectic solvents and electrolytes.

DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses

The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr–GPSGCC and SBDSco–GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA–protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.