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Inflammation and the Gram-positive anaerobic bacterium Cutibacterium acnes, which is implicated in acne pathogenesis and pilosebaceous unit inflammation, are the main targets of antibiotic-based therapy against acne vulgaris (acne). The most widely used antibiotics in acne therapy are tetracyclines, macrolides, and lincosamides. Unfortunately, C. acnes bacteria over the past several decades have demonstrated increased resistance to these antibiotics, particularly to clindamycin. The precise knowledge of how antibiotics interact with their clinical target is needed to overcome this problem. Toward this goal, we determined the structure of clindamycin in complex with the ribosome of Cutibacterium acnes at 2.53 Å resolution using cryogenic electron microscopy. The galactose sugar moiety of clindamycin interacts with nucleotides of the 23S rRNA directly or through a conserved network of water-mediated interactions. Its propyl pyrrolidinyl group interacts with the 23S rRNA through van der Waals forces. Clindamycin binding to the Cutibacterium acnes ribosome interferes with both: proper orientation of the aminoacyl group of the A-site bound tRNA that is needed for peptide bond formation and with the extension of the nascent peptide. Our data are important for advancing understanding of antibiotic resistance and development of narrow- spectrum antibacterial drugs, which is an urgent need for contemporary antibiotic stewardship.
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Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo-electron microscopy (cryo-EM). With this in situ cryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast to in vitro studies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolution in situ studies in the complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.
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All betacoronaviruses (ß-CoVs) encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts host gene expression. Among the ß-CoV family, MERS-CoV is the most distantly related member to SARS-CoV-2, and the mechanism for host translation inhibition by MERS-CoV Nsp1 remains controversial. Herein, we show that MERS-CoV Nsp1 directly interacts with the 40S ribosomal subunit. Using cryogenic electron microscopy (cryo-EM), we report a 2.6-Å structure of the MERS-CoV Nsp1 bound to the human 40S ribosomal subunit. The extensive interactions between C-terminal domain of MERS-CoV Nsp1 and the mRNA entry channel of the 40S ribosomal subunit are critical for its translation inhibition function. This mechanism of MERS-CoV Nsp1 is strikingly similar to SARS-CoV and SARS-CoV-2 Nsp1, despite modest sequence conservation. Our results reveal that the mechanism of host translation inhibition is conserved across ß-CoVs and highlight a potential therapeutic target for the development of antivirals that broadly restrict ß-CoVs.
Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , SARS-CoV-2/genética , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
The innate immune receptor RIG-I recognizes 5'-triphosphate double-stranded RNAs (5' PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5' PPP-end with m7G and methylate the 2'-O-ribose to evade RIG-I, preventing aberrant immune responses deleterious to the cell. Recent studies have identified RNAs in cells capped with metabolites such as NAD+, FAD and dephosphoCoA. Whether RIG-I recognizes these metabolite-capped RNAs has not been investigated. Here, we describe a strategy to make metabolite-capped RNAs free from 5' PPP dsRNA contamination, using in vitro transcription initiated with metabolites. Mechanistic studies show that metabolite-capped RNAs have a high affinity for RIG-I, stimulating the ATPase activity at comparable levels to 5' PPP dsRNA. Cellular signaling assays show that the metabolite-capped RNAs potently stimulate the innate antiviral immune response. This demonstrates that RIG-I can tolerate diphosphate-linked, capped RNAs with bulky groups at the 5' RNA end. This novel class of RNAs that stimulate RIG-I signaling may have cellular roles in activating the interferon response and may be exploited with proper functionalities for RIG-I-related RNA therapeutics.
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RNA Helicases DEAD-box , RNA de Cadeia Dupla , Proteína DEAD-box 58/genética , RNA Helicases DEAD-box/metabolismo , Imunidade Inata , Interferons/genética , Ligantes , Capuzes de RNA , RNA Viral/genética , RNA Viral/metabolismo , Transdução de Sinais , HumanosRESUMO
Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.
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Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , HIV-1/metabolismo , Transporte Ativo do Núcleo Celular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Infecções por HIV/metabolismo , Poro Nuclear/metabolismoRESUMO
Acne vulgaris is a chronic disfiguring skin disease affecting â¼1 billion people worldwide, often having persistent negative effects on physical and mental health. The Gram-positive anaerobe, Cutibacterium acnes is implicated in acne pathogenesis and is, therefore, a main target for antibiotic-based acne therapy. We determined a 2.8-Å resolution structure of the 70S ribosome of Cutibacterium acnes by cryogenic electron microscopy and discovered that sarecycline, a narrow-spectrum antibiotic against Cutibacterium acnes, may inhibit two active sites of this bacterium's ribosome in contrast to the one site detected previously on the model ribosome of Thermus thermophilus. Apart from the canonical binding site at the mRNA decoding center, the second binding site for sarecycline exists at the nascent peptide exit tunnel, reminiscent of the macrolides class of antibiotics. The structure also revealed Cutibacterium acnes-specific features of the ribosomal RNA and proteins. Unlike the ribosome of the Gram-negative bacterium Escherichia coli, Cutibacterium acnes ribosome has two additional proteins, bS22 and bL37, which are also present in the ribosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We show that bS22 and bL37 have antimicrobial properties and may be involved in maintaining the healthy homeostasis of the human skin microbiome.
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Acne Vulgar , Antibacterianos , Propionibacterium acnes , Ribossomos , Tetraciclinas , Humanos , Acne Vulgar/tratamento farmacológico , Acne Vulgar/microbiologia , Antibacterianos/química , Propionibacterium acnes/efeitos dos fármacos , Biossíntese de Proteínas , Ribossomos/efeitos dos fármacos , Tetraciclinas/farmacologiaRESUMO
Group II introns are ribozymes that catalyze their self-excision and function as retroelements that invade DNA. As retrotransposons, group II introns form ribonucleoprotein (RNP) complexes that roam the genome, integrating by reversal of forward splicing. Here we show that retrotransposition is achieved by a tertiary complex between a structurally elaborate ribozyme, its protein mobility factor, and a structured DNA substrate. We solved cryo-electron microscopy structures of an intact group IIC intron-maturase retroelement that was poised for integration into a DNA stem-loop motif. By visualizing the RNP before and after DNA targeting, we show that it is primed for attack and fits perfectly with its DNA target. This study reveals design principles of a prototypical retroelement and reinforces the hypothesis that group II introns are ancient elements of genetic diversification.
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Íntrons , Splicing de RNA , RNA Catalítico , Retroelementos , Ribonucleoproteínas , Microscopia Crioeletrônica , Ribonucleoproteínas/química , RNA Catalítico/química , DNA Polimerase Dirigida por RNA/genética , Eubacterium/enzimologia , Eubacterium/genéticaRESUMO
The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.
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RNA Helicases DEAD-box , RNA de Cadeia Dupla , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Imunidade Inata , Estrutura Terciária de Proteína , RNA Viral/genéticaRESUMO
The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for protein folding, lipid synthesis, and calcium homeostasis. Maintenance of ER structural integrity is crucial for proper function, but much remains to be learned about the molecular players involved. To identify proteins that support the structure of the ER, we performed a proteomic screen and identified nodal modulator (NOMO), a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We found that overexpression of NOMO1 imposes a sheet morphology on the ER, whereas depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network positive for the lysosomal marker lysosomal-associated protein 1. In addition, the levels of key players of autophagy including microtubule-associated protein light chain 3 and autophagy cargo receptor p62/sequestosome 1 strongly increase upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a "beads on a string" structure likely representing consecutive immunoglobulin-like domains. Extending NOMO1 by insertion of additional immunoglobulin folds results in a correlative increase in the ER intermembrane distance. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.
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Retículo Endoplasmático , Proteômica , Proteína Sequestossoma-1 , Autofagia , Estresse do Retículo Endoplasmático , Homeostase , Humanos , Lisossomos/metabolismoRESUMO
The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.
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COVID-19/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Animais , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/virologia , SARS-CoV-2/genética , SARS-CoV-2/ultraestrutura , Células Vero , Proteínas não Estruturais Virais/genéticaRESUMO
The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of proteins functions in the innate immune system. The A3 proteins are interferon inducible and hypermutate deoxycytidine to deoxyuridine in foreign single-stranded DNA (ssDNA). However, this deaminase activity cannot discriminate between foreign and host ssDNA at the biochemical level, which presents a significant danger when A3 proteins gain access to the nucleus. Interestingly, this A3 capability can be harnessed when coupled with novel CRISPR-Cas9 proteins to create a targeted base editor. Specifically, A3A has been used in vitro to revert mutations associated with disease states. Recent structural studies have shown the importance of loop regions of A3A and A3G in ssDNA recognition and positioning for deamination. In this work, we further examined loop 1 of A3A to determine how it affects substrate selection, as well as the efficiency of deamination, in the hopes of advancing the potential of A3A in base editing technology. We found that mutating residue H29 enhanced deamination activity without changing substrate specificity. Also interestingly, we found that increasing the length of loop 1 decreases substrate specificity. Overall, these results lead to a better understanding of substrate recognition and deamination by A3A and the A3 family of proteins.
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Citidina Desaminase/química , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Citidina Desaminase/genética , DNA de Cadeia Simples/genética , Desaminação/fisiologia , Humanos , Mutação/fisiologia , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Proteínas/genéticaRESUMO
Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N1-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.
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Autoimunidade/genética , Proteína DEAD-box 58/genética , Imunidade Inata/genética , Capuzes de RNA/imunologia , RNA de Cadeia Dupla/imunologia , Adenosina Trifosfatases/metabolismo , Domínio de Ativação e Recrutamento de Caspases/imunologia , Proteína DEAD-box 58/química , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Medição da Troca de Deutério/métodos , Mutação com Ganho de Função , Guanosina/análogos & derivados , Guanosina/química , Guanosina/imunologia , Guanosina/metabolismo , Helicase IFIH1 Induzida por Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Espectrometria de Massas/métodos , Metilação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Ligação Proteica/imunologia , Capuzes de RNA/química , Capuzes de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Viral/imunologia , Receptores Imunológicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
RIG-I has a remarkable ability to specifically select viral 5'ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient-state kinetics, we elucidated the ATPase-driven "kinetic proofreading" mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off rates. ATP binding facilitates dsRNA engagement but, interestingly, makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merten syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5'ppp dsRNA but, more importantly, drives RIG-I oligomerization through translocation, which we show to be regulated by helicase motif IVa. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5'ppp end "throttles" translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.
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Adenosina Trifosfatases/metabolismo , Proteína DEAD-box 58/metabolismo , RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Doenças da Aorta/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Hipoplasia do Esmalte Dentário/metabolismo , Feminino , Células HEK293 , Humanos , Hidrólise , Cinética , Metacarpo/anormalidades , Metacarpo/metabolismo , Doenças Musculares/metabolismo , Odontodisplasia/metabolismo , Osteoporose/metabolismo , Ligação Proteica/fisiologia , RNA de Cadeia Dupla/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Imunológicos , Ribossomos/metabolismo , Transdução de Sinais/fisiologia , Calcificação Vascular/metabolismoRESUMO
RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl (2'-OMe) group to the 5'-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5'ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2'-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2'-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution.
Assuntos
Guanosina/análogos & derivados , Imunidade Inata , Capuzes de RNA/metabolismo , RNA Helicases/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Guanosina/química , Guanosina/metabolismo , Células HEK293 , Humanos , Hidrólise , Metilação , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA/química , RNA de Cadeia Dupla , Transdução de SinaisRESUMO
RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5' triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5'-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a 'gate' that prevents cellular RNAs from generating productive complexes that can signal.