Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA, a rate-limiting step in fatty acid biosynthesis
. 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 β-oxidation
. ACC1 is regulated by a complex interplay of phosphorylation, binding of allosteric regulators and protein-protein interactions, which is further linked to filament formation
. These filaments were discovered in vitro and in vivo 50 years ago
, 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.
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.
HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival ...factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal ∼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.
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•Structure of a full-length HECT ligase reveals modular assembly•HUWE1 folds into ring-shaped architecture with flexibly attached accessory domains•Patient mutations interfere with HUWE1 ligase activity•Armadillo repeats in the ring architecture contribute to substrate binding
Hunkeler et al. present the cryo-EM structure of HUWE1, a large HECT E3 ligase that forms a modular ring-shaped assembly with flexibly attached accessory domains. The influence of mutations associated with intellectual disabilities on HUWE1 activity and substrate recognition by HUWE1 is dissected biochemically and structurally.
Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in ...eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.
HUWE1 is a large, enigmatic HECT-domain ubiquitin ligase implicated in the regulation of diverse pathways, including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally ...diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we find that HUWE1 targets substrates in a largely cell-type-specific manner. However, we identify C16orf72/HAPSTR1 as a robust HUWE1 substrate in multiple cell lines. Previously established physical and genetic interactions between HUWE1 and HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is required for HUWE1 nuclear localization and nuclear substrate targeting. Nuclear HUWE1 is required for both cell proliferation and modulation of stress signaling pathways, including p53 and nuclear factor κB (NF-κB)-mediated signaling. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.
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•HUWE1 substrates are diverse and largely cell-type specific•HAPSTR1/C16orf72 is a HUWE1 substrate in multiple cell lines•HAPSTR1 localizes HUWE1 to the nucleus•Nuclear HUWE1 is required for cell proliferation and modulates p53 and NF-κB pathways
Monda et al. find that the substrates of the ubiquitin ligase, HUWE1, are diverse and largely cell-type specific. HAPSTR1 is a robust substrate of HUWE1 and is required for nuclear localization of HUWE1. Nuclear HUWE1 is required for cell proliferation and modulation of stress signaling pathways.
Secretion of proteins into the membrane-cell wall space is essential for cell wall biosynthesis and pathogenicity in Gram-positive bacteria. Folding and maturation of many secreted proteins depend on ...a single extracellular foldase, the PrsA protein. PrsA is a 30-kDa protein, lipid anchored to the outer leaflet of the cell membrane. The crystal structure of Bacillus subtilis PrsA reveals a central catalytic parvulin-type prolyl isomerase domain, which is inserted into a larger composite NC domain formed by the N- and C-terminal regions. This domain architecture resembles, despite a lack of sequence conservation, both trigger factor, a ribosome-binding bacterial chaperone, and SurA, a periplasmic chaperone in Gram-negative bacteria. Two main structural differences are observed in that the N-terminal arm of PrsA is substantially shortened relative to the trigger factor and SurA and in that PrsA is found to dimerize in a unique fashion via its NC domain. Dimerization leads to a large, bowl-shaped crevice, which might be involved in vivo in protecting substrate proteins from aggregation. NMR experiments reveal a direct, dynamic interaction of both the parvulin and the NC domain with secretion propeptides, which have been implicated in substrate targeting to PrsA.
Background: PrsA is a foldase for secreted proteins and pathogenicity factors in Gram-positive bacteria.
Results: Crystallographic, enzymatic, and NMR spectroscopic analysis provide insights to PrsA function.
Conclusion: Substrate peptides interact around a unique crevice generated by PrsA dimerization via its chaperone-like domain.
Significance: The comprehensive characterization of PrsA promotes its utilization as foldase and drug target.
Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis, and inhibitor of apoptosis ...proteins (IAPs) are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived proapoptotic factors such as SMAC and HTRA2. Through a series of cryo-electron microscopy structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to SMAC, caspases, and HTRA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by SMAC. The architecture of BIRC6, together with near-irreversible binding of SMAC, elucidates how the IAP inhibitor SMAC can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.
Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy ...of drug-induced degradation of transcription factors and other cancer targets
, but a substantial subset of proteins are resistant to targeted degradation using existing approaches
. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL6
. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.
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.
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•Crystal structures of core domains of a single-chain bacterial carboxylase•Analysis of large-scale domain motions in the 680-kDa carboxylase•Hybrid model integrating crystallographic and solution scattering data•Paradigm for the dynamic architecture of carrier protein-based multienzymes
YCCs are a family of biotin-dependent multienzyme carboxylases. Combining X-ray crystallography, electron microscopy, and solution X-ray scattering, Hagmann et al. obtained a hybrid model of a 680-kDa hexameric bacterial YCC and a quantitative description of its remarkably dynamic protein architecture.
BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6’s broad-complex, ...tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.
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•18 ZBTB TFs show evidence of polymerization in cellular fluorescent reporter assay•Cryo-EM structures reveal that ZBTB5BTB and ZBTB9BTB polymerize into filaments•Polymerization enhances chromatin occupancy at homotypic clusters of binding sites•Polymerization augments transcriptional repression by ZBTB TFs
Park, Park et al. identify a subset of transcription factors (TFs) that polymerize through their BTB domains. Self-assembly of these ZBTB TFs enhances chromatin occupancy at regions containing homotypic clusters of binding motifs and, in turn, augments repression of target genes.
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