ABSTRACT
Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating activity. Recent studies have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments. Here we describe an ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). Cryo-EM structures reveal that CTPS2 filaments dynamically switch between active and inactive forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures reveal a link between conformation and control of ammonia channeling between the enzyme's active sites, and explain differences in regulation of human CTPS isoforms. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.
Subject(s)
Carbon-Nitrogen Ligases/chemistry , Protein Multimerization , Carbon-Nitrogen Ligases/metabolism , Carbon-Nitrogen Ligases/ultrastructure , Catalytic Domain , Cryoelectron Microscopy , Enzyme Activation , Humans , Models, Molecular , Protein Conformation , Substrate SpecificityABSTRACT
A rich and ongoing history of cell biology research has defined the major polymer systems of the eukaryotic cytoskeleton. Recent studies have identified additional proteins that form filamentous structures in cells and can self-assemble into linear polymers when purified. This suggests that the eukaryotic cytoskeleton is an even more complex system than previously considered. In this essay, I examine the case for an expanded definition of the eukaryotic cytoskeleton and present a series of challenges for future work in this area.
Subject(s)
Cytoskeleton/ultrastructure , Drosophila melanogaster/chemistry , Eukaryotic Cells/ultrastructure , Schizosaccharomyces/chemistry , Actins/chemistry , Actins/ultrastructure , Animals , Carbon-Nitrogen Ligases/chemistry , Carbon-Nitrogen Ligases/ultrastructure , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/ultrastructure , Cytoskeleton/chemistry , Drosophila melanogaster/cytology , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/ultrastructure , Endosomes/chemistry , Endosomes/ultrastructure , Escherichia coli/chemistry , Escherichia coli/cytology , Eukaryotic Cells/chemistry , Humans , Protein Multimerization , Protein Transport , Schizosaccharomyces/cytology , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/ultrastructureABSTRACT
Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).
