RESUMEN
RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.
Asunto(s)
Bacteriófagos , Bacteriófagos/metabolismo , Microscopía por Crioelectrón/métodos , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfato , Adenosina Desaminasa/metabolismoRESUMEN
The NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here, we report that an endogenous, stimulus-responsive form of full-length mouse NLRP3 is a 12- to 16-mer double-ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for many NLRP3-activating stimuli, requires the double-ring cages of NLRP3. Double-ring-defective NLRP3 mutants abolish inflammasome punctum formation, caspase-1 processing, and cell death. Thus, our data uncover a physiological NLRP3 oligomer on the membrane that is poised to sense diverse signals to induce inflammasome activation.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Quinasas Relacionadas con NIMA/genética , Proteína con Dominio Pirina 3 de la Familia NLR/aislamiento & purificación , Proteína con Dominio Pirina 3 de la Familia NLR/ultraestructura , Nigericina/farmacología , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Red trans-Golgi/metabolismoRESUMEN
Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Esteroles/metabolismo , Proteínas de Transporte Vesicular/química , Transporte Biológico , Microscopía por Crioelectrón , Cristalografía , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Dominios Proteicos , Vacuolas/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.
RESUMEN
Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a ß-barrel structure1,2. ß-Barrels are sheets of ß-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3-5. One subunit of the machines is itself a ß-barrel protein that has a central role in folding other ß-barrels. In Gram-negative bacteria, the ß-barrel assembly machine (BAM) consists of the ß-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid ß-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/química , Cloroplastos/química , Proteínas de Escherichia coli/química , Bacterias Gramnegativas/química , Enlace de Hidrógeno , Mitocondrias/química , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Proteasome inhibitors are widely used anticancer drugs. The three clinically approved agents are modified small peptides that preferentially target one of the proteasome's three active sites (ß5) at physiologic concentrations. In addition to these drugs, there is also an endogenous proteasome inhibitor, PI31/Fub1, that enters the proteasome's interior to simultaneously yet specifically inhibit all three active sites. Here, we have used PI31's evolutionarily optimized inhibitory mechanisms to develop a suite of potent and specific ß2 inhibitors. The lead compound strongly inhibited growth of multiple myeloma cells as a standalone agent, indicating the compound's cell permeability and establishing ß2 as a potential therapeutic target in multiple myeloma. The lead compound also showed strong synergy with the existing ß5 inhibitor bortezomib; such combination therapies might help with existing challenges of resistance and severe side effects. These results represent an effective method for rational structure-guided development of proteasome inhibitors.
Asunto(s)
Antineoplásicos , Mieloma Múltiple , Humanos , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/química , Bortezomib/farmacología , Bortezomib/uso terapéuticoRESUMEN
The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of a low-density lipoprotein receptor class A (LDLa) module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. In the present study, we determine the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.
Asunto(s)
Relaxina , Humanos , Relaxina/química , Relaxina/metabolismo , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/químicaRESUMEN
RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
Asunto(s)
Proteínas 14-3-3/antagonistas & inhibidores , Proteínas 14-3-3/química , Microscopía por Crioelectrón , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sitios de Unión , Transformación Celular Neoplásica/genética , Humanos , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Much of cellular activity is mediated by large multisubunit complexes. However, many of these complexes are too complicated to assemble spontaneously. Instead, their biogenesis is facilitated by dedicated chaperone proteins, which are themselves excluded from the final product. This is the case for the proteasome, a ubiquitous and highly conserved cellular regulator that mediates most selective intracellular protein degradation in eukaryotes. The proteasome consists of two subcomplexes: the core particle (CP), where proteolysis occurs, and the regulatory particle (RP), which controls substrate access to the CP. Ten chaperones function in proteasome biogenesis. Here, we review the pathway of CP biogenesis, which requires five of these chaperones and proceeds through a highly ordered multistep pathway. We focus on recent advances in our understanding of CP assembly, with an emphasis on structural insights. This pathway of CP biogenesis represents one of the most dramatic examples of chaperone-mediated assembly and provides a paradigm for understanding how large multisubunit complexes can be produced.
Asunto(s)
Chaperonas Moleculares , Complejo de la Endopetidasa Proteasomal , Eucariontes/metabolismo , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ProteolisisRESUMEN
The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure-activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at â¼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding.
Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Arabidopsis/enzimología , Inhibidores Enzimáticos/química , Hidroliasas/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Arabidopsis/química , Arabidopsis/efectos de los fármacos , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Herbicidas/química , Hidroliasas/química , Hidroliasas/ultraestructura , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.
Asunto(s)
Microscopía por Crioelectrón/métodos , Membrana Dobles de Lípidos/química , Maleatos/química , Proteínas de la Membrana/química , Poliestirenos/química , Cristalografía por Rayos X , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/ultraestructura , Conformación Proteica , Multimerización de Proteína , Proteolípidos/química , Proteolípidos/ultraestructuraRESUMEN
Membrane proteins are ubiquitous in biology and are key targets for therapeutic development. Despite this, our structural understanding has lagged behind that of their soluble counterparts. This review provides an overview of this important field, focusing in particular on the recent resurgence of electron microscopy (EM) and the increasing role it has to play in the structural studies of membrane proteins, and illustrating this through several case studies. In addition, we examine some of the challenges remaining in structural determination, and what steps are underway to enhance our knowledge of these enigmatic proteins.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.
Asunto(s)
Proteínas de Escherichia coli/química , Membrana Dobles de Lípidos/química , Maleatos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Poliestirenos/química , Proteínas Recombinantes/química , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Microscopía Electrónica/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Coloración y Etiquetado/métodosRESUMEN
The rotary ATPase family comprises the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and archaeal ATPase (A-ATPase). These either predominantly utilize a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in EM has come a significant increase in our understanding of the rotary ATPase family. Following the sub nm resolution reconstructions of both the F- and V-ATPases, the secondary structure organization of the elusive subunit a has now been resolved, revealing a novel helical arrangement. Despite these significant developments in our understanding of the rotary ATPases, there are still a number of unresolved questions about the mechanism, regulation and overall architecture, which this mini-review aims to highlight and discuss.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Eucariontes/metabolismoRESUMEN
The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.
Asunto(s)
Albúminas/metabolismo , Insecticidas/metabolismo , Pisum sativum/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Albúminas/antagonistas & inhibidores , Secuencia de Bases , Benzofenonas/metabolismo , Sitios de Unión , Biotina/metabolismo , Cartilla de ADN , Insecticidas/química , Microscopía Electrónica , Etiquetas de Fotoafinidad , Unión Proteica , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD(+) bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD(+) and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhibitory activity at 1 µM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.
Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Sistemas de Liberación de Medicamentos , Enoil-ACP Reductasa (NADH)/antagonistas & inhibidores , Toxoplasma/efectos de los fármacos , Antiparasitarios/química , Antiparasitarios/farmacología , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.
Asunto(s)
Microscopía por Crioelectrón , Modelos Moleculares , Chaperonas Moleculares , Complejo de la Endopetidasa Proteasomal , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Conformación ProteicaRESUMEN
Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.
RESUMEN
Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the ß5 and ß2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.