Structure and dynamics of the active site gorge of acetylcholinesterase: Synergistic use of molecular dynamics simulation and X‐ray crystallography

The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 Å from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis‐quaternary decamethonium (DECA) ion, acquired during enzyme purification, resides in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.

[1]  G. Rose,et al.  Loops in globular proteins: a novel category of secondary structure. , 1986, Science.

[2]  G A Petsko,et al.  Mapping protein dynamics by X-ray diffraction. , 1985, Progress in biophysics and molecular biology.

[3]  J. Sussman,et al.  Relationship between sequence conservation and three‐dimensional structure in a large family of esterases, lipases, and related proteins , 1993, Protein science : a publication of the Protein Society.

[4]  H. Tilbeurgh,et al.  Interfacial activation of the lipase–procolipase complex by mixed micelles revealed by X-ray crystallography , 1993, Nature.

[5]  J. Sussman,et al.  An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[6]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.

[7]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[8]  J. Sussman,et al.  Acetylcholinesterase: Structure and use as a model for specific cation-protein interactions , 1992, Current Biology.

[9]  F. Hucho,et al.  Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N‐dimethyl‐2‐phenyl‐aziridinium. , 1990, The EMBO journal.

[10]  M. Karplus,et al.  Crystallographic R Factor Refinement by Molecular Dynamics , 1987, Science.

[11]  M. L. Connolly.  Solvent-accessible surfaces of proteins and nucleic acids. , 1983, Science.

[12]  J L Sussman,et al.  Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C. , 1988, Journal of molecular biology.

[13]  H. Berman,et al.  Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography. , 1986, The Journal of biological chemistry.

[14]  P. Axelsen,et al.  Molecular dynamics of tryptophan in ribonuclease-T1. I. Simulation strategies and fluorescence anisotropy decay. , 1988, Biophysical Journal.

[15]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[16]  E. Gratton,et al.  Experimentally verifying molecular dynamics simulations through fluorescence anisotropy measurements. , 1991, Biochemistry.

[17]  J. Sussman,et al.  Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Karplus,et al.  Stochastic boundary conditions for molecular dynamics simulations of ST2 water , 1984 .

[19]  P. Axelsen,et al.  Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence. , 1989, Biophysical journal.

[20]  A. Goldman,et al.  Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein , 1991, Science.

[21]  J. Sussman,et al.  Differential effects of "peripheral" site ligands on Torpedo and chicken acetylcholinesterase. , 1994, Molecular pharmacology.

[22]  J. Mccammon,et al.  Molecular dynamics with stochastic boundary conditions , 1982 .

[23]  J L Sussman,et al.  Acetylcholinesterase: electrostatic steering increases the rate of ligand binding. , 1993, Biochemistry.

[24]  D. Quinn,et al.  Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states , 1987 .

[25]  M. Karplus,et al.  Active site dynamics in protein molecules: A stochastic boundary molecular‐dynamics approach , 1985, Biopolymers.

[26]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[27]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[28]  J L Sussman,et al.  Open "back door" in a molecular dynamics simulation of acetylcholinesterase. , 1994, Science.