João M. Damas

Structural consequences of ATP hydrolysis on the ABC transporter NBD dimer: molecular dynamics studies of HlyB.

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.

The multicopper oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity

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.

Predicting the Thermodynamics and Kinetics of Helix Formation in a Cyclic Peptide Model

The peptide Ac-(cyclo-2,6)-R[KAAAD]-NH2 (cyc-RKAAAD) is a short cyclic peptide known to adopt a remarkably stable single turn α-helix in water. Due to its simplicity and the availability of thermodynamic and kinetic experimental data, cyc-RKAAAD poses as an ideal model for evaluating the aptness of current molecular dynamics (MD) simulation methodologies to accurately sample conformations that reproduce experimentally observed properties. In this work, we extensively sample the conformational space of cyc-RKAAAD using microsecond-timescale MD simulations. We characterize the peptide conformational preferences in terms of secondary structure propensities and, using Cartesian-coordinate principal component analysis (cPCA), construct its free energy landscape, thus obtaining a detailed weighted discrimination between the helical and nonhelical subensembles. The cPCA state discrimination, together with a Markov model built from it, allowed us to estimate the free energy of unfolding (-0.57 kJ/mol) and the relaxation time (∼0.435 μs) at 298.15 K, which are in excellent agreement with the experimentally reported values (-0.22 kJ/mol and 0.42 μs, Serrano, A. L.; Tucker, M. J.; Gai, F. J. Phys. Chem. B, 2011, 115, 7472-7478.). Additionally, we present simulations conducted using two enhanced sampling methods: replica-exchange molecular dynamics (REMD) and bias-exchange metadynamics (BE-MetaD). We compare the free energy landscape obtained by these two methods with the results from MD simulations and discuss the sampling and computational gains achieved. Overall, the results obtained attest to the suitability of modern simulation methods to explore the conformational behavior of peptide systems with a high level of realism.

Exploring O2 Diffusion in A-Type Cytochrome c Oxidases: Molecular Dynamics Simulations Uncover Two Alternative Channels towards the Binuclear Site

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.

Putative Dioxygen-Binding Sites and Recognition of Tigecycline and Minocycline in the Tetracycline-Degrading Monooxygenase Tetx

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 Pathway for O2 Diffusion inside CotA Laccase and Possible Implications on the Multicopper Oxidases Family.

Laccases and multicopper oxidases (MCOs) oxidize a wide range of organic compounds while reducing O2 to water, enabling numerous biotechnological applications. It is still unknown how O2 reaches the internalized catalytic center of MCOs where it gets reduced, despite a proposed channel inferred from X-ray crystallography structures. Herein, an alternative new pathway is found through the use of a combination of free energy calculations (implicit ligand sampling), landscape analysis, and Markov modeling. The reported pathway is shown to be the one mostly contributing to O2 reaching the catalytic center. This pathway is considered in light of the whole MCO family, and a relation to the protonation state of a structurally conserved acidic residue right above the center is advanced.

High-Throughput Automated Preparation and Simulation of Membrane Proteins with HTMD

HTMD is a programmable scientific platform intended to facilitate simulation-based research in molecular systems. This paper presents the functionalities of HTMD for the preparation of a molecular dynamics simulation starting from PDB structures, building the system using well-known force fields, and applying standardized protocols for running the simulations. We demonstrate the frameworks flexibility for high-throughput molecular simulations by applying a preparation, building, and simulation protocol with multiple force-fields on all of the seven hundred eukaryotic membrane proteins resolved to-date from the orientation of proteins in membranes (OPM) database. All of the systems are available on www.playmolecule.org .

MD Simulations Reveal an Alternative Pathway for Dioxygen Diffusion in Aa3 Cytochrome C Oxidases

Cytochrome c oxidases (CCOX) are members of the heme-copper oxidase superfamily and they are the terminal enzymes of the respiratory chain. These proteins are membrane-bound multi-subunit redox-driven proton pumps, which couple the reduction of molecular dioxygen to water with the creation of a transmembrane electrochemical proton gradient.Over the last 20 years, most of the CCOX research focused on the mechanisms and energetics of reduction and/or proton pumping and little emphasis has been given to the pathways used by dioxygen to reach the binuclear site. The main objective of this work is to identify possible alternative dioxygen pathways in the reduced CCOX from Rhodobacter sphaeroides [1] using extensive Molecular Dynamics (MD) simulations. Our simulations allowed the identification of two possible dioxygen channels, whose entrances are both located in the membrane region. The first channel is a Y-shaped hydrophobic cavity with a constriction point near F282(I) and W172(I), and it corresponds to the oxygen pathway previously identified in the X-ray structure [2]. The second channel follows the hydroxylfarnesyl tail of haem a3 and ends near the Y288(I) (which is covalently linked to the H284(I) imidazole group).[1] Qin et al. (2009) Biochemistry 48, 5121-5130.[2] Svensson-EK et al. (2002) J.Mol.Biol.321, 329-339.