Our continuing investigation of acetylcholinesterase (AChE), the acetylcholine binding protein (AChBP) reflect a long standing and continuous commitment to the study of proteins that affect the intensity and duration of acetylcholine action, a critical neurotransmitter affecting cognition in the CNS and peripheral autonomic and motor function. Our studies, supported by this grant over the past 37 years, have evolved from considerations of structure and function of AChE and the nicotinic acetylcholine receptor (nAChR) and, more recently, from AChE to a related a/b-hydrolase fold protein, neuroligin. In turn, they have spun off separate drug development endeavors directed novel nicotinic receptor ligands for depression, schizophrenia, pain alleviation and nicotine addiction and AChE inhibitor antidotes. Our fundamental studies with the extracellular domain of the three proteins are now based on structure at atomic resolution, that of static crystal structures, but also extend to an analysis of conformational dynamics and assignments of energetic contributions through structural modification and mutant cycle analysis. Conformation and dynamics are examined through fluorescence spectroscopy, decay of fluorescence anisotropy and H/D exchange. Our nicotinic receptor studies focus on an interfacial site between subunits that can be examined by physical methods through the soluble receptor surrogate, AChBP. Crystal structures of the complexes selected from a wide array of ligands with AChBP enable a detailed analysis of the structural determinants of specificity. We propose to expand to presumed non-competitive sites on this molecule, in particular the vestibule leading into the channel constriction and the non-a subunit interfaces that do not bind agonist. With AChE, we propose to continue our analysis of complexes formed by freeze-frame, click chemistry to compare complexes dictated by kinetics of association and by achieving thermodynamic equilibrium. We also will examine the inductive role of the oxyanion hole, the nucleophile rendering catalytic triad and the hydrogen bonding network between the proximal serine hydroxyls at the base of the gorge. Studies with the heterophilic adhesion protein, neuroligin (NL), capitalize on its homologous structure to AChE, both being members of the a/b-hydrolase-fold family, wherein their common globular domains and unique recognition features help direct the study of NL by low angle scattering and high resolution techniques. This approach is helping to uncover the molecular determinants associated with the interaction of NL with its synaptic partner proteins at its structurally unique binding site. Moreover, the common structural fold between AChE and NL allows comparisons in how mutations affect biosynthesis and trafficking of these two molecules. The shared familial structural features enable us to understand how gene mutations, some of which are associated with autism spectrum disorders, affect the biosynthesis and folding in this protein family.

Public Health Relevance

Our proposed research is directed to understanding the structure and function of three proteins, acetylcholinesterase, the nicotinic acetylcholine receptor and neuroligin, that are related to each other in terms of structural homology and coordination of neurotransmitter function. Since these proteins are found at synapses in the nervous system and mediate signaling events and synaptic architecture, they are important potential drug targets and indicators of genetic predisposition to certain disorders in the nervous system. Our structural and functional studies, using X ray crystallography and solution-based spectroscopic and spectrometric techniques, offer new avenues into understanding the roles of these three proteins in physiological function and therapeutic outcomes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM018360-41
Application #
8385536
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Dunsmore, Sarah
Project Start
1975-01-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
41
Fiscal Year
2013
Total Cost
$687,969
Indirect Cost
$242,682
Name
University of California San Diego
Department
None
Type
Schools of Pharmacy
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Morris, Garrett M; Green, Luke G; Radic, Zoran et al. (2013) Automated docking with protein flexibility in the design of femtomolar "click chemistry" inhibitors of acetylcholinesterase. J Chem Inf Model 53:898-906
Radic, Zoran; Sit, Rakesh K; Garcia, Edzna et al. (2013) Mechanism of interaction of novel uncharged, centrally active reactivators with OP-hAChE conjugates. Chem Biol Interact 203:67-71
Taylor, Palmer; De Jaco, Antonella; Comoletti, Davide et al. (2013) Cholinesterase confabs and cousins: approaching forty years. Chem Biol Interact 203:10-3
Rodriguez, Laura P; Vilarino, Natalia; Molgo, Jordi et al. (2013) Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system. Anal Chem 85:2340-7
De Jaco, Antonella; Comoletti, Davide; Dubi, Noga et al. (2012) Processing of cholinesterase-like ?/?-hydrolase fold proteins: alterations associated with congenital disorders. Protein Pept Lett 19:173-9
Grimster, Neil P; Stump, Bernhard; Fotsing, Joseph R et al. (2012) Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template. J Am Chem Soc 134:6732-40
Nemecz, Akos; Taylor, Palmer (2011) Creating an ?7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses. J Biol Chem 286:42555-65
Yamauchi, John G; Nemecz, Ákos; Nguyen, Quoc Thang et al. (2011) Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors. PLoS One 6:e16519
Valle, Anne M; Radic, Zoran; Rana, Brinda K et al. (2011) Naturally occurring variations in the human cholinesterase genes: heritability and association with cardiovascular and metabolic traits. J Pharmacol Exp Ther 338:125-33
Miller, Meghan T; Mileni, Mauro; Comoletti, Davide et al. (2011) The crystal structure of the ?-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function. Structure 19:767-78

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