ABSTRACT
Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA, a rate-limiting step in fatty acid biosynthesis1,2. Eukaryotic acetyl-CoA carboxylases are large, homodimeric multienzymes. Human acetyl-CoA carboxylase occurs in two isoforms: the metabolic, cytosolic ACC1, and ACC2, which is anchored to the outer mitochondrial membrane and controls fatty acid ß-oxidation1,3. ACC1 is regulated by a complex interplay of phosphorylation, binding of allosteric regulators and protein-protein interactions, which is further linked to filament formation1,4-8. These filaments were discovered in vitro and in vivo 50 years ago7,9,10, but the structural basis of ACC1 polymerization and regulation remains unknown. Here, we identify distinct activated and inhibited ACC1 filament forms. We obtained cryo-electron microscopy structures of an activated filament that is allosterically induced by citrate (ACC-citrate), and an inactivated filament form that results from binding of the BRCT domains of the breast cancer type 1 susceptibility protein (BRCA1). While non-polymeric ACC1 is highly dynamic, filament formation locks ACC1 into different catalytically competent or incompetent conformational states. This unique mechanism of enzyme regulation via large-scale conformational changes observed in ACC1 has potential uses in engineering of switchable biosynthetic systems. Dissecting the regulation of acetyl-CoA carboxylase opens new paths towards counteracting upregulation of fatty acid biosynthesis in disease.
Subject(s)
Acetyl-CoA Carboxylase/chemistry , Acetyl-CoA Carboxylase/ultrastructure , Cryoelectron Microscopy , Acetyl-CoA Carboxylase/metabolism , Animals , BRCA1 Protein/chemistry , BRCA1 Protein/pharmacology , Biopolymers/chemistry , Biopolymers/metabolism , Cell Line , Citric Acid/pharmacology , Humans , Models, Molecular , Polymerization/drug effects , Protein Domains/drug effects , Protein Structure, Quaternary/drug effects , Spodoptera , Structure-Activity RelationshipABSTRACT
Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes.
Subject(s)
Acetyl-CoA Carboxylase/chemistry , Bacterial Proteins/chemistry , Biotin/chemistry , Carbon-Nitrogen Ligases/chemistry , Carboxyl and Carbamoyl Transferases/chemistry , Deinococcus/chemistry , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Amino Acid Motifs , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Baculoviridae/genetics , Baculoviridae/metabolism , Binding Sites , Biotin/metabolism , Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/metabolism , Carboxyl and Carbamoyl Transferases/genetics , Carboxyl and Carbamoyl Transferases/metabolism , Cloning, Molecular , Crystallography, X-Ray , Deinococcus/enzymology , Escherichia coli/chemistry , Escherichia coli/enzymology , Fatty Acid Synthase, Type II/chemistry , Fatty Acid Synthase, Type II/genetics , Fatty Acid Synthase, Type II/metabolism , Gene Expression , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , Sf9 Cells , Spodoptera , Substrate Specificity , X-Ray DiffractionABSTRACT
Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.
