Robert C. Rittenhouse

Coordination number of zinc ions in the phosphotriesterase active site by molecular dynamics and quantum mechanics.

We have run several molecular dynamics (MD) simulations on zinc‐containing phosphotriesterase (PTE) with two bound substrates, sarin and paraoxon, and with the substrate analog diethyl 4‐methylbenzylphosphonate. A standard nonbonded model was employed to treat the zinc ions with the commonly used charge of +2. In all the trajectories, we observed a tightly bound water (TBW) molecule in the active site that was coordinated to the less buried zinc ion. The phosphoryl oxygen of the substrate/inhibitor was found to be coordinated to the same zinc ion so that, considering all ligands, the less buried zinc was hexa‐coordinated. The hexa‐coordination of this zinc ion was not seen in the deposited X‐ray pdb files for PTE. Several additional MD simulations were then performed using different charges (+1, +1.5) on the zinc ions, along with ab initio and density functional theory (DFT) calculations, to evaluate the following possibilities: the crystal diffraction data were not correctly interpreted; the hexa‐coordinated zinc ion in PTE is only present in solution and not in the crystal; and the hexa‐coordinated zinc ion in PTE is an artifact of the force field used. A charge of +1.5 leads to a coordination number (CN) of 5 on both zinc ions, which is consistent with the results from ab initio and DFT calculations and with the latest high resolution X‐ray crystal structure. The commonly used charge of +2 produces a CN of 6 on the less buried zinc. The CN on the more buried zinc ion is 5 when the substrate/inhibitor is present in the simulation, and increases to 6 when the substrate/inhibitor is removed prior to the simulation. The results of both of the MD and quantum mechanical calculations lead to the conclusion that the zinc ions in the PTE active site are both penta‐coordinated, and that the MD simulations performed with the charge of +2 overestimate the CN of the zinc ions in the PTE active site. The overall protein structures in the simulations remain unaffected by the change in zinc charge from +2 to +1.5. The results also suggest that the charge +1.5 is the most appropriate for the molecular dynamics simulations on zinc‐containing PTE when a nonbonded model is used and no global thermodynamic conclusion is sought. We also show that the standard nonbonded model is not able to properly treat the CN and energy at the same time. A preliminary, promising charge‐transfer model is discussed with the use of the zinc charge of +1.5.

Characterization of the active site of DNA polymerase β by molecular dynamics and quantum chemical calculation

It is well established that the fully formed polymerase active site of the DNA repair enzyme, polymerase β (pol β), including two bound Mg2+ cations and the nucleoside triphosphate (dNTP) substrate, exists at only one point in the catalytic cycle just prior to the chemical nucleotidyl transfer step. The structure of the active conformation has been the subject of much interest as it relates to the mechanism of the chemical step and also to the question of fidelity assurance. Although crystal structures of ternary pol β–(primer‐template) DNA–dNTP complexes have provided the main structural features of the active site, they are necessarily incomplete due to intentional alterations (e.g., removal of the 3′OH groups from primer and substrate) needed to obtain a structure from midcycle. Working from the crystal structure closest to the fully formed active site [Protein Data Bank (PDB) code: 1bpy], two molecular dynamics (MD) simulations of the solvated ternary complex were performed: one with the missing 3′OHs restored, via modeling, to the primer and substrate, and the other without restoration of the 3′OHs. The results of the simulations, together with ab initio optimizations on simplified active‐site models, indicate that the missing primer 3′OH in the crystal structure is responsible for a significant perturbation in the coordination sphere of the catalytic cation and allow us to suggest several corrections and additions to the active‐site structure as observed by crystallography. In addition, the calculations help to resolve questions raised regarding the protonation states of coordinating ligands. Proteins 2003;53:000–000.

Rutherford: Exploring the scattering of alpha particles

Abstracts for Volume VB, Number 2. This simulation permits students to design and implement scattering experiments of the sort performed by Rutherford, Geiger, and Marsden.