The mechanism of substrate release by the aspartate transporter GltPh: insights from simulations.

Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, Glt(Ph), has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of Glt(Ph). In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na(+) to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate-protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na(+), the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.

Origins of the regioselectivities in the Diels-Alder reactions of vinylindenes with 1,4-quinone monoketal and acrolein dienophiles.

Computations with density functional theory (B3LYP/6-31G(d)) have elucidated the origins of regioselectivities in the Diels-Alder reaction of vinylindene with a 1,4-quinone monoketal reaction that was employed as the key step in the synthesis of fluostatin C. Frontier Molecular Orbital theory and an electrostatic model are applied to the reactions of alkyl-substituted and vinylindene dienes with 1,4-quinone monoketal and acrolein dienophiles. Regiochemical results that deviate from expectation are explained by a progression from electronic to steric control upon the addition of a Lewis acid catalyst.

Kemp elimination catalysts by computational enzyme design.

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.

Quantum mechanical design of enzyme active sites.

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

How similar are enzyme active site geometries derived from quantum mechanical theozymes to crystal structures of enzyme-inhibitor complexes? Implications for enzyme design.

Quantum mechanical optimizations of theoretical enzymes (theozymes), which are predicted catalytic arrays of biological functionalities stabilizing a transition state, have been carried out for a set of nine diverse enzyme active sites. For each enzyme, the theozyme for the rate-determining transition state plus the catalytic groups modeled by side-chain mimics was optimized using B3LYP/6-31G(d) or, in one case, HF/3-21G(d) quantum mechanical calculations. To determine if the theozyme can reproduce the natural evolutionary catalytic geometry, the positions of optimized catalytic atoms, i.e., covalent, partial covalent, or stabilizing interactions with transition state atoms, are compared to the positions of the atoms in the X-ray crystal structure with a bound inhibitor. These structure comparisons are contrasted to computed substrate-active site structures surrounded by the same theozyme residues. The theozyme/transition structure is shown to predict geometries of active sites with an average RMSD of 0.64 A from the crystal structure, while the RMSD for the bound intermediate complexes are significantly higher at 1.42 A. The implications for computational enzyme design are discussed.

The origins of femtomolar protein-ligand binding: hydrogen-bond cooperativity and desolvation energetics in the biotin-(strept)avidin binding site.

The unusually strong reversible binding of biotin by avidin and streptavidin has been investigated by density functional and MP2 ab initio quantum mechanical methods. The solvation of biotin by water has also been studied through QM/MM/MC calculations. The ureido moiety of biotin in the bound state hydrogen bonds to five residues, three to the carbonyl oxygen and one for each--NH group. These five hydrogen bonds act cooperatively, leading to stabilization that is larger than the sum of individual hydrogen-bonding energies. The charged aspartate is the key residue that provides the driving force for cooperativity in the hydrogen-bonding network for both avidin and streptavidin by greatly polarizing the urea of biotin. If the residue is removed, the network is disrupted, and the attenuation of the energetic contributions from the neighboring residues results in significant reduction of cooperative interactions. Aspartate is directly hydrogen-bonded with biotin in streptavidin and is one residue removed in avidin. The hydrogen-bonding groups in streptavidin are computed to give larger cooperative hydrogen-bonding effects than avidin. However, the net gain in electrostatic binding energy is predicted to favor the avidin-bicyclic urea complex due to the relatively large penalty for desolvation of the streptavidin binding site (specifically expulsion of bound water molecules). QM/MM/MC calculations involving biotin and the ureido moiety in aqueous solution, featuring PDDG/PM3, show that water interactions with the bicyclic urea are much weaker than (strept)avidin interactions due to relatively low polarization of the urea group in water.

Density functional theory determination of an axial gateway to explain the rate and endo selectivity enhancement of Diels-Alder reactions by bis(oxazoline)-Cu(II).

A novel conceptual model and unique understanding of rate and endo selectivity enhancements delivered by bis(oxazoline)-Cu(II) Lewis acid catalysts in the Diels-Alder reaction of cyclopentadiene and acrylate imide is presented. Despite previously reported kinetic and spectroscopic studies, the physical reasons for endo selectivity and rate enhancements remain poorly understood. Large-scale density functional calculations using Becke three-parameter density functional theory with the nonlocal correlation of Lee, Yang, and Parr and the 6-31G(d) basis set have been carried out for the first time to understand the geometric and energetic consequences of C(2)-substituent variation. The unique positioning of the tert-butyl C(2)-substituent with respect to the diene, referred to as the "axial gateway", maintains the electrophilicity of the catalyst by shielding the reactive metal center from nucleophilic attack. The interplay between steric and electronic factors is crucial to understanding the observed enhanced rates and endo selectivity.

Enhanced sampling by multiple molecular dynamics trajectories: carbonmonoxy myoglobin 10 micros A0-->A(1-3) transition from ten 400 picosecond simulations.

The utility of multiple trajectories to extend the time scale of molecular dynamics simulations is reported for the spectroscopic A-states of carbonmonoxy myoglobin (MbCO). Experimentally, the A0-->A(1-3) transition has been observed to be 10 micros at 300 K, which is beyond the time scale of standard molecular dynamics simulations. To simulate this transition, 10 short (400 ps) and two longer time (1.2 ns) molecular dynamics trajectories, starting from five different crystallographic and solution phase structures with random initial velocities centered in a 37 A radius sphere of water, have been used to sample the native-fold of MbCO. Analysis of the ensemble of structures gathered over the cumulative 5.6 ns reveals two biomolecular motions involving the side chains of His64 and Arg45 to explain the spectroscopic states of MbCO. The 10 micros A0-->A(1-3) transition involves the motion of His64, where distance between His64 and CO is found to vary up to 8.8 +/- 1.0 A during the transition of His64 from the ligand (A(1-3)) to bulk solvent (A0). The His64 motion occurs within a single trajectory only once, however the multiple trajectories populate the spectroscopic A-states fully. Consequently, multiple independent molecular dynamics simulations have been found to extend biomolecular motion from 5 ns of total simulation to experimental phenomena on the microsecond time scale.