Anandamide Hydrolysis in FAAH Reveals a Dual Strategy for Efficient Enzyme-Assisted Amide Bond Cleavage via Nitrogen Inversion

Herein, we combined classical molecular dynamics (MD) and quantum mechanical/molecular mechanics (QM/MM) simulations to unravel the whole catalytic cycle of fatty acid amide hydrolase (FAAH) in complex with anandamide, the main neurotransmitters involved in the control of pain. While microsecond MD simulations of FAAH in a realistic membrane/water environment provided a solid model for the reactant state of the enzymatic complex (Palermo et al. J. Chem. Theory Comput. 2013, 9, 12021213.), QM/MM simulations depict now a highly concerted two-step catalytic mechanism characterized by (1) acyl-enzyme formation after hydrolysis of the substrate amide bond and (2) deacylation reaction with restoration of the catalytic machinery. We found that a crucial event for anandamide hydrolysis is the inversion of the reactive nitrogen of the scissile amide bond, which occurs during the acylation rate-limiting step. We show that FAAH uses an exquisite catalytic strategy to induce amide bond distortion, reactive nitrogen inversion, and amide bond hydrolysis, promoting catalysis to completion. This new strategy is likely to be of general applicability to other amidases/peptidases that show similar catalytic site architectures, providing crucial insights for de novo enzyme design or drug discovery efforts.

Characterization of the Dizinc Analogue of the Synthetic Diiron Protein DF1 Using ab Initio and Hybrid Quantum/Classical Molecular Dynamics Simulations

Ursula Röthlisberger

The structural and dynamical properties of the four a-helix bundle Due Ferri 1, which is a generic mimic of diiron proteins, have been explored using d. functional theory (DFT) based and hybrid quantum/classical mol. dynamics (QM/MM) simulations. Four quantum mech. (QM) representations of the active site have been employed in order to systematically assess the role of first and second shell interactions: (a) a 66-atom fragment of the active site comprising only first shell ligands, (b and c) two systems (78 and 86 atoms) contg. different second shell hydrogen bonds, and (d) a 98-atom model including both the first and second shell residues. Two QM/MM partitioning schemes have been explored in order to explicitly consider the role of the whole protein environment: (a) a 54-QM-atom model in which only the first shell ligands are described at the DFT level, while the rest of the protein environment is taken into account at the MM level and (b) a 68-QM-atom model in which the first shell ligands plus a second shell hydrogen bond network is considered at the DFT level. All of the calcns. confirm the highly flexible nature of the carboxylate-bridged binuclear motif and demonstrate the importance of the whole protein environment in stabilizing the hydrogen bond networks that surround the active site. The present QM/MM approach allows for the identification of key factors governing the stability/reactivity of the active site and thus provides unique insights that can be exploited for the future tailoring of new highly selective biomimetic enzymic compds. [on SciFinder (R)]

2003

On the structural stability of the free and metal-loaded c-terminal domain of the prion protein

Maria Carola Colombo

A misfolded conformer of the cellular prion protein, denoted as scrapie prion protein, is considered responsible for a variety of fatal neurodegenerative diseases. Both, the function of the protein in its native conformation as well as the factors that trigger the protein misfolding and its mechanism remain as yet open issues. The aim of this thesis is to give a contribution to answering these questions by means of a variety of tools currently available in the computational (bio)chemistry community. The capability of the cellular prion protein isoform to bind metal is supported by controversial evidences and atomistic details of putative binding sites are missing. Based on the NMR structure of the C-terminal part of the cellular isoform, we performed a hybrid quantum/classical (QM/MM) Car-Parrinello molecular dynamics study to identify likely Cu(II) binding sites. By means of a bioinformatics approach, we localized the protein regions with the highest propensity for copper ion binding. The identified candidate structures were subsequently refined via QM/MM simulations and then probed by computing their EPR characteristics. Overall best agreement with the experimental EPR data was observed for a binding site involving H187. The previously characterized prion protein Cu(II)-binding sites were substituted for Mn(II) and Zn(II) and subsequently refined by means of the QM/MM Car-Parrinello approach. As a general conclusion, all the Cu-binding sites lead to stable arrangements upon Mn and Zn substitution with relative minor structural changes even within the ps time scale. The validity of the binding motifs obtained was assessed by means of comparison with a pool of known Mn and Zn binding proteins and common features were found and described. The Zn binding site in proximity of H177 was speculated to act as a protein interface zinc site with a possible direct involvement in the aggregation process. The EPR parameters of the Mn-containing binding sites were also computed. The influence of some environmental factors that can possibly trigger the misfolding event were investigated via classical molecular dynamics simulations. Specifically, protein solution containing a monovalent salt (NaCl) in four different concentrations (0.0, 0.05, 0.1, 0.2 M) were simulated at three different pH (pH 2, 4, 7) in order to highlight structural changes and local weaknesses in the protein native structural motifs. The effects of the ionic strength and pH on the secondary structural elements of the protein were identified and characterized whereas the main topological framework was preserved in all simulations. The energy landscape of the cellular to scrapie isoform misfolding pathway is currently under investigation by means of a parallel tempering (replica exchange) enhanced sampling technique in combination with classical molecular dynamics. The screening of the data collected led to the identification of a pool of prion protein scrapie candidates. A set of new folding motifs were characterized. Preliminary results and plans for future investigations are presented. With the goal of providing a structural model for the minimal misfolded aggregate based on the most likely prion protein scrapie candidates, a protein-protein docking approach was used to generate protofibril models and their structural stability and transformations investigated by means of classical molecular dynamics simulations.

EPFL2006

An Atomistic Simulation Method for Oxygen Impurities in Aluminium Based on Variable Charge Molecular Dynamics

Andreas Elsener

Impurities are known to have a significant impact on materials properties. In particular, the presence of impurities can change mechanical properties and stabilize the microstructure by reducing grain growth and recrystallization processes. In the past atomistic simulations, in particular molecular dynamics, have contributed a lot to the understanding of deformation mechanisms in nanocrystalline metals. Especially, insights into details of dislocation/GB interactions have been gained. Additionally, simulations on coupled grain boundary migration in bicrystalline samples have played an important role in understanding stress assisted grain growth in nanocrystalline metals. However, all these simulations have been performed on monatomic systems. This means that the role of impurities has not yet been considered in these simulations. Some of the important difference between experiments and simulations could be removed by introducing dilute O distributions to computational Al samples. For this purpose, a simulation method is required which can deal with the oxidation of Al atoms around the O impurities and which can simulate the ionic and metallic nature of bonding simultaneously. Here, a method is suggested which fulfills the requirements by modifying the variable charge method of Streitz and Mintmire [Phys. Rev. B 50, 11996 (1994)]. A local chemical potential approach which optimizes the charge on only those atoms expected to be ionic is developed. Additionally the EAM potential for the non-electrostatic interaction given in the publication of Streitz and Mintmire is substituted by empirical potentials which treat the Al-Al interactions with a well-known Al potential. The Al-O and O-O interactions in these EAM potentials are fitted for the simulation of dilute O impurities in Al and additionally for the simulation of corundum in case of a second potential. The aforementioned developments are applied to study the effect of O impurities on the deformation of nc Al. A fully three-dimensional nc sample containing 15 grains of 12 nm grain-size is deformed with a dilute O content in the triple junction lines. The impact of O impurities on dislocation propagation is also considered in a sample which consists of a single grain extracted from a large molecular dynamics simulation. This simulation geometry contains a perfect dislocation, which is pinned at the surrounding grain boundary network. Therefore it allows the study of pinning strength and after unpinning the propagation behavior of the dislocation under various O impurity distributions. Additionally, the role of O impurities on coupled GB migration is investigated by studying two bicrystalline samples with different O distributions in both of them. In the discussion, the emphasis is on the simulation method. Especially, the significance of the samples, simulation conditions and the performance of the method in the different applications are discussed. Additionally, the different developments (local chemical potential approach and EAM potentials) are critically analyzed and improvements for future simulations of impurities are suggested.