ABC transporters are a large and important family of membrane proteins involved in substrate transport across the membrane. The transported substrates are quite diverse, ranging from monatomic ions to large biomolecules. Consequently, some ABC transporters are involved in biomedically relevant situations, from genetic diseases to multidrug resistance. The most conserved domains in ABC transporters are the nucleotide binding domains (NBDs), which form a dimer responsible for the binding and hydrolysis of ATP, concomitantly with substrate translocation. To elucidate how ATP hydrolysis structurally affects the NBD dimer, and consequently the transporter, we performed a molecular dynamics study on the NBD dimer of the HlyB ABC exporter. We have observed a change in the contact surface between the monomers after hydrolysis, even though we have not seen dimer opening in any of the five 100 ns simulations. We have also identified specific regions that respond to ATP hydrolysis, in particular the X-loop motif of ABC exporters, which has been shown to be in contact with the coupling helices of the transmembrane domains (TMDs). We propose that this motif is an important part of the NBD-TMD communication in ABC exporters. Through nonequilibrium analysis, we have also identified gradual conformational changes within a short time scale after ATP hydrolysis.

Fast and accurate molecular force field (FF) parameterization is still an unsolved problem. Accurate FFs are not generally available for all molecules, like novel druglike molecules. While methods based on quantum mechanics (QM) exist to parameterize them with better accuracy, they are computationally expensive and slow, which limits applicability to a small number of molecules. Here, we present an automated FF parameterization method which can utilize either DFT calculations or approximate arXiv:1907.06952v2 [physics.chem-ph] 3 Aug 2019 QM energies produced by different neural network potentials (NNPs), to obtain improved parameters for molecules. We demonstrate that for the case of torchani-ANI-1x NNP, we can parameterize small molecules in a fraction of time compared with an equivalent parameterization using DFT QM calculations while producing more accurate parameters than FF (GAFF2). We expect our method to be of critical importance in computational structure-based drug discovery. The current version is available at PlayMolecule (www.playmolecule.org) and implemented in HTMD, allowing to parameterize molecules with different QM and NNP options.

Expression of the aromatic hydroxylase TetX under aerobic conditions confers bacterial resistance against tetracycline antibiotics. Hydroxylation inactivates and degrades tetracyclines, preventing inhibition of the prokaryotic ribosome. X-ray crystal structure analyses of TetX in complex with the second-generation and third-generation tetracyclines minocycline and tigecycline at 2.18 and 2.30 Å resolution, respectively, explain why both clinically potent antibiotics are suitable substrates. Both tetracyclines bind in a large tunnel-shaped active site in close contact to the cofactor FAD, pre-oriented for regioselective hydroxylation to 11a-hydroxytetracyclines. The characteristic bulky 9-tert-butylglycylamido substituent of tigecycline is solvent-exposed and does not interfere with TetX binding. In the TetX-minocycline complex a second binding site for a minocycline dimer is observed close to the active-site entrance. The pocket is formed by the crystal packing arrangement on the surface of two neighbouring TetX monomers. Crystal structure analysis at 2.73 Å resolution of xenon-pressurized TetX identified two adjacent Xe-binding sites. These putative dioxygen-binding cavities are located in the substrate-binding domain next to the active site. Molecular-dynamics simulations were performed in order to characterize dioxygen-diffusion pathways to FADH2 at the active site.

The multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV–visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the multicopper oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site‐directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 °C, a half‐life of inactivation of ∼ 6 h at 80 °C, and temperature values at the midpoint from 97 to 112 °C. McoP is an efficient metallo‐oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min−1, respectively, at 40 °C. It is noteworthy that McoP follows a ping‐pong mechanism, with three‐fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of multicopper oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.

Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.