9rt3 | pdb_00009rt3

Structure of Choline O-acetyltransferase in complex with (E)-4-(2-((2-acetamidoethyl)thio)-2-(naphthalen-1-yl)vinyl)-1-methylpyridin-1-ium

spinqualitylabelsall models 9rt3, resolution 1.80Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 9rt3 is a 1 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.8Å Ligands: A1JJI, EDO, NA, PEG Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Disease CLAT_HUMAN Defects in CHAT are the cause of congenital myasthenic syndrome with episodic apnea (CMSEA) [MIM:254210; formerly known as familial infantile myasthenia gravis 2 (FIMG2). CMSEA is an autosomal recessive congenital myasthenic syndrome. Patients have myasthenic symptoms since birth or early infancy, negative tests for anti-AChR antibodies, and abrupt episodic crises with increased weakness, bulbar paralysis, and apnea precipitated by undue exertion, fever, or excitement. Function CLAT_HUMAN Catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Publication Abstract from PubMed Choline O-acetyltransferase (ChAT) catalyzes the biosynthesis of acetylcholine and is a cysteine-rich enzyme that has been investigated using a range of biochemical and structural approaches. Existing ChAT ligands rely on electrophilic or unstable scaffolds that limit their suitability for biological systems. Prior work established that arylvinylpyridiniums (AVPs) are substrate mimics that undergo ChAT-catalyzed hydrothiolation with CoA to form covalent AVP-CoA adducts. Here, we applied a structure-guided strategy to design non-reactive ligands intended to mimic key features of the AVP-CoA binding pose while avoiding covalent reactivity. Nineteen analogs were synthesized and evaluated across complementary biochemical, structural, and biophysical assays. X-ray crystallography confirmed that the new ligands bind within the ChAT tunnel similar to AVP-CoAs. Importantly, the high cysteine content of ChAT, especially within a reactive CXCXXC motif, rendered the enzyme susceptible to modification by the widely used 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) reagent used for measuring ChAT activity, leading to confounding results in thiol-dependent activity assays. Enzyme-free counter-screens demonstrated that all apparent inhibitory activity arose from interference with assay readout rather than true enzymatic inhibition. Surface plasmon resonance measurements established that none of the designed ligands display detectable reversible affinity for ChAT, despite their crystallographically validated poses, and no selectivity over the related enzyme carnitine-O-acetyltransferase (CrAT) was observed. These findings demonstrate that confirmed binding with X-ray crystallography is insufficient to confer functional interaction with ChAT and highlight the susceptibility of this enzyme to thiol-reactive assay artefacts. More broadly, this work underscores the necessity of integrating orthogonal biophysical validation when studying ligand binding to cysteine-rich enzymes. Analysis of ligand recognition by choline-O-acetyltransferase reveals thiol-reactive assay interference and weak ligand affinity in solution.,Forsgren N, Jonsson F, Carlsson M, Sander RA, Larsson A, Linden P, Linusson A, Engdahl CS, Wiktelius D, Ekstrom F J Biol Chem. 2026 May 6:113115. doi: 10.1016/j.jbc.2026.113115. PMID:42103233 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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