Abstract
Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.
| Original language | English |
|---|---|
| Article number | 101007 |
| Journal | Journal of Biological Chemistry |
| Volume | 297 |
| Issue number | 3 |
| DOIs | |
| State | Published - Sep 1 2021 |
Funding
Acknowledgments—The highly professional and creative assistance of the Stanford Synchrotron Radiation Lightsource (SSRL) BioSAXS beamline (BL 4-2) scientists Dr Thomas Weiss, Dr Tsutomu Mat-sui, and Dr Ivan Rajkovićis highly appreciated. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH and the NIGMS, National Institutes of Health (P41GM103393). We are grateful to Dr Jonah Cheung, NYSBC, New York, for providing us with atomic coordinates of PDB ID 5HF5 well before their deposition to the PDB. We thank Dina Schneidman-Duhovny ([email protected]) for her assistance in using MultiFoXS. This research was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), the National Institute of Neurological Disorders and Stroke (NINDS) (grant numbers U01 NS083451 and R21 NS098998), and the UCSD Academic Senate grant BG084144. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of General Medical Sciences or National Institutes of Health. The highly professional and creative assistance of the Stanford Synchrotron Radiation Lightsource (SSRL) BioSAXS beamline (BL 4-2) scientists Dr Thomas Weiss, Dr Tsutomu Matsui, and Dr Ivan Rajkovic is highly appreciated. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DEAC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH and the NIGMS, National Institutes of Health (P41GM103393). We are grateful to Dr Jonah Cheung, NYSBC, New York, for providing us with atomic coordinates of PDB ID 5HF5 well before their deposition to the PDB. We thank Dina Schneidman-Duhovny ([email protected]) for her assistance in using MultiFoXS. This research was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), the National Institute of Neurological Disorders and Stroke (NINDS) (grant numbers U01 NS083451 and R21 NS098998), and the UCSD Academic Senate grant BG084144. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of General Medical Sciences or National Institutes of Health.