RESUMO
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.
RESUMO
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.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Chaperonas Moleculares , Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Conformação ProteicaRESUMO
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.
Assuntos
Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Modelos Moleculares , Domínio Catalítico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteólise , CatáliseRESUMO
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.
RESUMO
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.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/química , Bortezomib/farmacologia , Bortezomib/uso terapêuticoRESUMO
PP2A serine/threonine protein phosphatases are heterotrimeric complexes that have a wide range of essential physiologic functions. The B55α form of PP2A has critical roles in cell cycle regulation, mitotic exit, and the DNA damage response1-6. Its activity is modulated by additional regulatory proteins, such as ARPP197, FAM122A8, and IER59. However, the precise mechanisms underlying the modulation of PP2A activity by these proteins remain elusive. Here, we show that IER5 inhibits pTau dephosphorylation by PP2A/B55α in biochemical assays and report a cryoelectron microscopy structure of the PP2A/B55α-IER5 complex, which reveals that IER5 occludes a surface on B55α used for substrate recruitment10-12. Mutation of interface residues on IER5 interferes with recovery of B55α in co-immunoprecipitation assays and suppresses events in squamous carcinoma cells, such as KRT1 expression, that depend on inhibition of PP2A/B55α by IER59. These studies define the molecular basis for PP2A inhibition by IER5 and suggest a roadmap for selective pharmacologic modulation of PP2A/B55α complexes.
RESUMO
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 level. 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 (AT1R) and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to AT1R 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.
RESUMO
Assembly of the proteasome's core particle (CP), a barrel-shaped chamber of four stacked rings, requires five chaperones and five subunit propeptides. Fusion of two half-CP precursors yields a complete structure but remains immature until active site maturation. Here, using Saccharomyces cerevisiae, we report a high-resolution cryogenic electron microscopy structure of preholoproteasome, a post-fusion assembly intermediate. Our data reveal how CP midline-spanning interactions induce local changes in structure, facilitating maturation. Unexpectedly, we find that cleavage may not be sufficient for propeptide release, as residual interactions with chaperones such as Ump1 hold them in place. We evaluated previous models proposing that dynamic conformational changes in chaperones drive CP fusion and autocatalytic activation by comparing preholoproteasome to pre-fusion intermediates. Instead, the data suggest a scaffolding role for the chaperones Ump1 and Pba1/Pba2. Our data clarify key aspects of CP assembly, suggest that undiscovered mechanisms exist to explain CP fusion/activation, and have relevance for diseases of defective CP biogenesis.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Complexo de Endopeptidases do Proteassoma/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Chaperonas MolecularesRESUMO
OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.
Assuntos
Interleucina-2 , Canais Iônicos , Animais , Interleucina-2/genética , Interleucina-2/metabolismo , Canais Iônicos/metabolismo , Mutação/genéticaRESUMO
RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.
Assuntos
Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas p21(ras) , Microscopia Crioeletrônica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Membrana Celular , Sistema de Sinalização das MAP QuinasesRESUMO
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.
Assuntos
Relaxina , Humanos , Relaxina/química , Relaxina/metabolismo , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/químicaRESUMO
Adhesion G Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to effect cell signalling. We report here that ADGRF1 can signal through all major G protein classes and identify the structural basis for a previously reported Gαq preference by cryo-EM. Our structure shows that Gαq preference in ADGRF1 may derive from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helix VII and helix VIII at the site of Gα recruitment. Mutational studies of the interface and of contact residues within the 7TM domain identify residues critical for signalling, and suggest that Gαs signalling is more sensitive to mutation of TA or binding site residues than Gαq. Our work advances the detailed molecular understanding of aGPCR TA activation, identifying features that potentially explain preferential signal modulation.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo , Sítios de Ligação , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Domínios Proteicos , Ligação ProteicaRESUMO
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.
Assuntos
Bacteriófagos , Bacteriófagos/metabolismo , Microscopia Crioeletrônica/métodos , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina , Adenosina Desaminase/metabolismoRESUMO
The Harvard Cryo-Electron Microscopy Center for Structural Biology, which was formed as a consortium between Harvard Medical School, Boston Children's Hospital, Dana-Farber Cancer Institute, and Massachusetts General Hospital, serves both academic and commercial users in the greater Harvard community. The facility strives to optimize research productivity while training users to become expert electron microscopists. These two tasks may be at odds and require careful balance to keep research projects moving forward while still allowing trainees to develop independence and expertise. This article presents the model developed at Harvard Medical School for running a research-oriented cryo-EM facility. Being a research-oriented facility begins with training in cryo-sample preparation on a trainee's own sample, ideally producing grids that can be screened and optimized on the Talos Arctica via multiple established pipelines. The first option, staff assisted screening, requires no user experience and a staff member provides instant feedback about the suitability of the sample for cryo-EM investigation and discusses potential strategies for sample optimization. Another option, rapid access, allows users short sessions to screen samples and introductory training for basic microscope operation. Once a sample reaches the stage where data collection is warranted, new users are trained on setting up data collection for themselves on either the Talos Arctica or Titan Krios microscope until independence is established. By providing incremental training and screening pipelines, the bottleneck of sample preparation can be overcome in parallel with developing skills as an electron microscopist. This approach allows for the development of expertise without hindering breakthroughs in key research areas.
RESUMO
Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome's barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Microscopia Crioeletrônica , Citoplasma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Chaperonas Moleculares , Complexo de Endopeptidases do Proteassoma , Eucariotos/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , ProteóliseRESUMO
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.
Assuntos
Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Human TMEM175, a noncanonical potassium (K+) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson's disease (PD). Structurally, the TMEM175 family exhibits an architecture distinct from canonical potassium channels, as it lacks the typical TVGYG selectivity filter. Here, we show that human TMEM175 not only exhibits pH-dependent structural changes that reduce K+ permeation at acidic pH but also displays proton permeation. TMEM175 constitutively conducts K+ at pH 7.4 but displays reduced K+ permeation at lower pH. In contrast, proton current through TMEM175 increases with decreasing pH because of the increased proton gradient. Molecular dynamics simulation, structure-based mutagenesis, and electrophysiological analysis suggest that K+ ions and protons share the same permeation pathway. The M393T variant of human TMEM175 associated with PD shows reduced function in both K+ and proton permeation. Together, our structural and electrophysiological analysis reveals a mechanism of TMEM175 regulation by pH.
RESUMO
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.