Accession Number:

ADA621684

Title:

A Structural Biology and Protein Engineering Approach to the Development of Antidotes Against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents

Descriptive Note:

Final rept. 11 Feb 2013-10 Feb 2015

Corporate Author:

WEIZMANN INST OF SCIENCE REHOVOT (ISRAEL)

Personal Author(s):

Report Date:

2015-05-01

Pagination or Media Count:

30.0

Abstract:

The human acetylcholinesterase hAChE gene was cloned into the pHLsec expression vector. The recombinant enzyme rhAChEmD was expressed on a large scale in adherent 293 tau cells. It was secreted as a monomeric species, purified by affinity chromatography, and deglycosylated with PNGase F. A crystallization screen, using the Mosquito crystallization robot, identified conditions for formation of diffraction-quality crystals in 0.025 dichloromethane12 PEG 20,0000.1M imidazole, pH 7.0. The crystals formed in the hexagonal space group P3112, with cell constants a125.31, b125.31, c131.40 A, and one monomer per asymmetric unit. A complete data set, to 2.9 A resolution, was collected at 100 K at the ESRF Grenoble, France. The structure was solved by molecular replacement, resulting in an Rfree of 23.74, and Rwork of 19.31, for all data to 2.9 A. The coordinates and structures factors have been deposited in the PDB, with IDcode 4PQE. As an initial step for synthesis of a GF surrogate, the chloridate, CH3POO-cyclohexylCl, has been synthesized, and will be reacted with coumarin. Synthesis of the pure chiral forms of the coumarin surrogate of VX, both the toxic Sp isomer, and the much less reactive and less toxic Rp form, in accordance with our published protocol, permitted preparation of the corresponding crystalline OPhAChE conjugates using the hAChE expressed in HEK293 cells, purified by affinity chromatography, and deglycosylated so as to produce well-diffracting crystals. The crystal structure of the Rp conjugate has been solved to 2.7 A, and reveals the interactions of the atoms of the OP moiety with amino-acid side-chains at atomic resolution. Conditions have also been developed for expression of a full-length rhAChE construct, rhAChEtau, which assembles to form the physiological tetramer.

Subject Categories:

  • Biochemistry
  • Anatomy and Physiology
  • Toxicology
  • Biomedical Instrumentation and Bioengineering

Distribution Statement:

APPROVED FOR PUBLIC RELEASE