Accurate pKa Calculations in Proteins with Reactive Molecular Dynamics Provide Physical Insight Into the Electrostatic Origins of Their Values.

Classical molecular dynamics simulations are a versatile tool in the study of biomolecular systems, but they usually rely on a fixed bonding topology, precluding the explicit simulation of chemical reactivity. Certain modifications can permit the modeling of reactions. One such method, multiscale reactive molecular dynamics, makes use of a linear combination approach to describe condensed-phase free energy surfaces of reactive processes of biological interest. Before these simulations can be performed, models of the reactive moieties must first be parametrized using electronic structure data. A recent study demonstrated that gas-phase electronic structure data can be used to derive parameters for glutamate and lysine which reproduce experimental pKa values in both bulk water and the staphylococcal nuclease protein with remarkable accuracy and transferability between the water and protein environments. In this work, we first present a new model for aspartate derived in similar fashion and demonstrate that it too produces accurate pKa values in both bulk and protein contexts. We also describe a modification to the prior methodology, involving refitting some of the classical force field parameters to density functional theory calculations, which improves the transferability of the existing glutamate model. Finally and most importantly, this reactive molecular dynamics approach, based on rigorous statistical mechanics, allows one to specifically analyze the fundamental physical causes for the marked pKa shift of both aspartate and glutamate between bulk water and protein and also to demonstrate that local steric and electrostatic effects largely explain the observed differences.

Synthesis, Characterization, and Simulation of Four-Armed Megamolecules.

This paper describes the synthesis, characterization, and modeling of a series of molecules having four protein domains attached to a central core. The molecules were assembled with the "megamolecule" strategy, wherein enzymes react with their covalent inhibitors that are substituted on a linker. Three linkers were synthesized, where each had four oligo(ethylene glycol)-based arms terminated in a para-nitrophenyl phosphonate group that is a covalent inhibitor for cutinase. This enzyme is a serine hydrolase and reacts efficiently with the phosphonate to give a new ester linkage at the Ser-120 residue in the active site of the enzyme. Negative-stain transmission electron microscopy (TEM) images confirmed the architecture of the four-armed megamolecules. These cutinase tetramers were also characterized by X-ray crystallography, which confirmed the active-site serine-phosphonate linkage by electron-density maps. Molecular dynamics simulations of the tetracutinase megamolecules using three different force field setups were performed and compared with the TEM observations. Using the Amberff99SB-disp + pH7 force field, the two-dimensional projection distances of the megamolecules were found to agree with the measured dimensions from TEM. The study described here, which combines high-resolution characterization with molecular dynamics simulations, will lead to a comprehensive understanding of the molecular structures and dynamics for this new class of molecules.

Effects of the Metal Ion on the Mechanism of Phosphodiester Hydrolysis Catalyzed by Metal-Cyclen Complexes.

In this study, mechanisms of phosphodiester hydrolysis catalyzed by six di- and tetravalent metal-cyclen (M-C) complexes (Zn-C, Cu-C, Co-C, Ce-C, Zr-C and Ti-C) have been investigated using DFT calculations. The activities of these complexes were studied using three distinct mechanisms: (1) direct attack ( DA ), (2) catalyst-assisted ( CA ), and (3) water-assisted ( WA ). All divalent metal complexes (Zn-C, Cu-C and Co-C) coordinated to the BNPP substrate in a monodentate fashion and activated its scissile phosphoester bond. However, all tetravalent metal complexes (Ce-C, Zr-C, and Ti-C) interacted with BNPP in a bidentate manner and strengthened this bond. The DA mechanism was energetically the most feasible for all divalent M-C complexes, while the WA mechanism was favored by the tetravalent complexes, except Ce-C. The divalent complexes were found to be more reactive than their tetravalent counterparts. Zn-C catalyzed the hydrolysis with the lowest barrier among all M-C complexes, while Ti-C was the most reactive tetravalent complex. The activities of Ce-C and Zr-C, except Ti-C, were improved with an increase in the coordination number of the metal ion. The structural and mechanistic information provided in this study will be very helpful in the development of more efficient metal complexes for this critical reaction.

A PM7 dynamic residue-ligand interactions energy landscape of the BACE1 inhibitory pathway by hydroxyethylamine compounds. Part I: The flap closure process.

BACE1 is an enzyme of scientific interest because it participates in the progression of Alzheimer's disease. Hydroxyethylamines (HEAs) are a family of compounds which exhibit inhibitory activity toward BACE1 at a nanomolar level, favorable pharmacokinetic properties and oral bioavailability. The first step in the inhibition of BACE1 by HEAs consists of their entrance into the protease active site and the resultant conformational change in the protein, from Apo to closed form. These two conformations differ in the position of an antiparallel loop (called the flap) which covers the entrance to the catalytic site. For BACE1, closure of this flap is vital to its catalytic activity and to inhibition of the enzyme due to the new interactions thereby formed with the ligand. In the present study a dynamic energy landscape of residue-ligand interaction energies (ReLIE) measured for 112 amino acids in the BACE1 active site and its immediate vicinity during the closure of the flap induced by 8 HEAs of different inhibitory power is presented. A total of 6.272 million ReLIE calculations, based on the PM7 semiempirical method, provided a deep and quantitative view of the first step in the inhibition of the aspartyl protease. The information suggests that residues Asp93, Asp289, Thr292, Thr293, Asn294 and Arg296 are anchor points for the ligand, accounting for approximately 45% of the total protein-ligand interaction. Additionally, flap closure improved the BACE1-HEA interaction by around 25%. Furthermore, the inhibitory activity of HEAs could be related to the capacity of these ligands to form said anchor point interactions and maintain them over time: the lack of some of these anchor interactions delayed flap closure or impeded it completely, or even caused the flap to reopen. The methodology employed here could be used as a tool to evaluate future structural modifications which lead to improvements in the favorability and stability of BACE1-HEA ReLIEs, aiding in the design of better inhibitors.

A molecular dynamics study of the BACE1 conformational change from Apo to closed form induced by hydroxyethylamine derived compounds.

BACE1 is an aspartyl protease which is a therapeutic target for Alzheimer's disease (AD) because of its participation in the rate-limiting step in the production of Aß-peptide, the accumulation of which produces senile plaques and, in turn, the neurodegenerative effects associated with AD. The active site of this protease is composed in part by two aspartic residues (Asp93 and Asp289). Additionally, the catalytic site has been found to be covered by an antiparallel hairpin loop called the flap. The dynamics of this flap are fundamental to the catalytic function of the enzyme. When BACE1 is inactive (Apo), the flap adopts an open conformation, allowing a substrate or inhibitor to access the active site. Subsequent interaction with the ligand induces flap closure and the stabilization of the macromolecular complex. Further, the protonation state of the aspartic dyad is affected by the chemical nature of the species entering the active site, so that appropriate selection of protonation states for the ligand and the catalytic residues will permit the elucidation of the inhibitory pathway for BACE1. In the present study, comparative analysis of different combinations of protonation states for the BACE1-hydroxyethylamine (HEA) system is reported. HEAs are potent inhibitors of BACE1 with favorable pharmacological and kinetic properties, as well as oral bioavailability. The results of Molecular Dynamics (MD) simulations and population density calculations using 8 different parameters demonstrate that the LnAsp289 configuration (HEA with a neutral amine and the Asp289 residue protonated) is the only one which permits the expected conformational change in BACE1, from apo to closed form, after flap closure. Additionally, differences in their capacities to establish and maintain interactions with residues such as Asp93, Gly95, Thr133, Asp289, Gly291, and Asn294 during this step allow differentiation among the inhibitory activities of the HEAs. The results and methodology here reported will serve to elucidate the inhibitory pathway of other families of compounds that act as BACE1 inhibitors, as well as the design of better leader compounds for the treatment of AD.