VMD as a Platform for Interactive Small Molecule Preparation and Visualization in Quantum and Classical Simulations.

Modeling and simulation of small molecules such as drugs and biological cofactors have been both a major focus of computational chemistry for decades and a growing need among computational biophysicists who seek to investigate the interaction of different types of ligands with biomolecules. Of particular interest in this regard are quantum mechanical (QM) calculations that are used to more accurately describe such small molecules, which can be of heterogeneous structures and chemistry, either in purely QM calculations or in hybrid QM/molecular mechanics (MM) simulations. QM programs are also used to develop MM force field parameters for small molecules to be used along with established force fields for biomolecules in classical simulations. With this growing need in mind, here we report a set of software tools developed and closely integrated within the broadly used molecular visualization/analysis program, VMD, that allow the user to construct, modify, and parametrize small molecules and prepare them for QM, hybrid QM/MM, or classical simulations. The tools also provide interactive analysis and visualization capabilities in an easy-to-use and integrated environment. In this paper, we briefly report on these tools and their major features and capabilities, along with examples of how they can facilitate molecular research in computational biophysics that might be otherwise prohibitively complex.

Rapid parameterization of small molecules using the Force Field Toolkit.

The inability to rapidly generate accurate and robust parameters for novel chemical matter continues to severely limit the application of molecular dynamics simulations to many biological systems of interest, especially in fields such as drug discovery. Although the release of generalized versions of common classical force fields, for example, General Amber Force Field and CHARMM General Force Field, have posited guidelines for parameterization of small molecules, many technical challenges remain that have hampered their wide-scale extension. The Force Field Toolkit (ffTK), described herein, minimizes common barriers to ligand parameterization through algorithm and method development, automation of tedious and error-prone tasks, and graphical user interface design. Distributed as a VMD plugin, ffTK facilitates the traversal of a clear and organized workflow resulting in a complete set of CHARMM-compatible parameters. A variety of tools are provided to generate quantum mechanical target data, setup multidimensional optimization routines, and analyze parameter performance. Parameters developed for a small test set of molecules using ffTK were comparable to existing CGenFF parameters in their ability to reproduce experimentally measured values for pure-solvent properties (<15% error from experiment) and free energy of solvation (±0.5 kcal/mol from experiment).

O2 reactivity of flavoproteins: dynamic access of dioxygen to the active site and role of a H+ relay system in D-amino acid oxidase.

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O(2) in the protein framework of the flavoprotein D-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O(2) leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O(2) high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O(2) affinity. In this variant, the reactivity of the reduced enzyme with O(2) is decreased >or=100-fold and the turnover number approximately 1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H(+) from the product imino acid =NH(2)(+) group bound on the flavin Re-side to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.

Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics.

Structural biology of mammalian lipoxygenases: enzymatic consequences of targeted alterations of the protein structure.

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the pathogenesis of diseases with major health political relevance (bronchial asthma, atherosclerosis, cancer, and osteoporosis). The crystal structures of one mammalian lipoxygenase and of two plant isoenzymes have been solved and the structural bases of important enzyme properties (reaction specificity, membrane binding, and suicidal inactivation) have been investigated in the past. This review will briefly summarize our current understanding on the structural biology of the most important mammalian lipoxygenase isoforms and will also address selected mechanistic features of the lipoxygenase reaction.

Dual role of oxygen during lipoxygenase reactions.

Studying the oxygenation kinetics of (19R/S,5Z,8Z,11Z,14Z)-19-hydroxyeicosa-5,8,11,14-tetraenoic acid (19-OH-AA) by rabbit 15-lipoxygenase-1 we observed a pronounced oxygen dependence of the reaction rate, which was not apparent with arachidonic acid as substrate. Moreover, we found that peroxide-dependent activation of the lipoxygenase depended strongly on the oxygen concentration. These data can be described with a kinetic model that extends previous schemes of the lipoxygenase reaction in three essential aspects: (a) the product of 19-OH-AA oxygenation is a less effective lipoxygenase activator than (13S,9Z,11E)-13-hydroperoxyoctadeca-9,11-dienoic acid; (b) molecular dioxygen serves not only as a lipoxygenase substrate, but also impacts peroxide-dependent enzyme activation; (c) there is a leakage of radical intermediates from the catalytic cycle, which leads to the formation of inactive ferrous lipoxygenase. This enzyme inactivation can be reversed by another round of peroxide-dependent activation. Taken together our data indicate that both peroxide activation and the oxygen affinity of lipoxygenases depend strongly on the chemistry of the lipid substrate. These findings are of biological relevance as variations of the reaction conditions may turn the lipoxygenase reaction into an efficient source of free radicals.

Molecular dynamics investigation of primary photoinduced events in the activation of rhodopsin.

Retinal cis-trans isomerization and early relaxation steps have been studied in a 10-ns molecular dynamics simulation of a fully hydrated model of membrane-embedded rhodopsin. The isomerization, induced by transiently switching the potential energy function governing the C(11)==C(12) dihedral angle of retinal, completes within 150 fs and yields a strongly distorted retinal. The most significant conformational changes in the binding pocket are straightening of retinal's polyene chain and separation of its beta-ionone ring from Trp-265. In the following 500 ps, transition of 6s-cis to 6s-trans retinal and dramatic changes in the hydrogen bonding network of the binding pocket involving the counterion for the protonated Schiff base, Glu-113, occur. Furthermore, the energy initially stored internally in the distorted retinal is transformed into nonbonding interactions of retinal with its environment. During the following 10 ns, increased mobilities of some parts of the protein, such as the kinked regions of the helices, mainly helix VI, and the intracellular loop I2, were observed, as well as transient structural changes involving the conserved salt bridge between Glu-134 and Arg-135. These features prepare the protein for major structural transformations achieved later in the photocycle. Retinal's motion, in particular, can be compared to an opening turnstile freeing the way for the proposed rotation of helix VI. This was demonstrated by a steered molecular dynamics simulation in which an applied torque enforced the rotation of helix VI.