Acetyl-coenzyme A synthetases convert ATP, acetate, and coenzyme A (CoA) into acetyl-CoA, a central metabolite that fuels lipid biosynthesis and regulates protein and RNA acetylation. ACS enzymes contain N- and C-terminal domains that coordinate a two-step ping-pong mechanism involving sequential adenylation and thioester formation at the interdomain interface. How domain motions coordinate these chemical steps remains unclear. Here, we report single-particle cryo-electron microscopy structures of Schizosaccharomyces pombe ACSA captured in apo, pre-adenylation, intermediate, and product states. These structures reveal ligand-dependent reorganization of the C-terminal domain: apo and pre-adenylation forms display increased conformational heterogeneity, whereas intermediate- and product-bound states adopt ordered conformations compatible with catalysis. Structure-guided mutagenesis and in vitro activity assays, together with sequence conservation, support the functional importance and evolutionary conservation of the observed conformational transitions across ACS homologs. These findings establish a ligand-coupled interdomain rearrangement mechanism underlying catalysis by ACS enzymes and a structural framework for inhibitor development.
Meng Li, Ming-Yang Zhou, R. Marmorstein· Structure· 0 citations
N-terminal acetyltransferase A (NatA) catalyzes 40-57% of eukaryotic Nα-acetylation, a co-translational modification critical for protein stability, localization, and function, yet no validated small-molecule probe of NatA has been reported. Here, we describe the discovery and characterization of tideglusib (YD3139), a clinical-stage GSK-3β inhibitor, as the first small-molecule probe for NatA through a unprecedented catalysis-dependent mechanism. This dual-event mechanism was established through intact protein MS, rapid dilution assays, and substrate-independent IC50 profiling. Tideglusib binds allosterically to Saccharomyces cerevisiae NatA (yNatA), whereupon the CoA released during catalysis chemically modifies tideglusib to form a reversible covalent adduct, a new paradigm for acetyltransferase inhibition. Tideglusib was identified by screening of an in-house library (∼700 compounds) and confirmed by structural optimization of the thiadiazolidinedione scaffold, yielding IC50 values of 0.68-1.8 μM against yeast and human NatA with >20-fold selectivity over other NAT family members. Quantitative proteomics confirmed on-target suppression of NatA-mediated Nα-acetylation in yeast. These findings establish tideglusib as a mechanistic chemical probe for NatA, reveal NatA as a previously unrecognized molecular target for a promiscuous clinical-stage investigational compound, and introduce a conceptual framework for exploiting catalysis-dependent adduct formation as a new strategy for acetyltransferase inhibitor design.
Youchao Deng, Zhuojun Luo, Sarah M. Gardner et al.· ACS Chemical Biology· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
