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1.
Nature ; 603(7900): 343-347, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35236982

RESUMEN

CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5-7. Although Cas9 variants with greater discrimination against mismatches have been designed8-10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.


Asunto(s)
Sistemas CRISPR-Cas , Reparación de la Incompatibilidad de ADN , Edición Génica , ARN Guía de Kinetoplastida , Proteína 9 Asociada a CRISPR/genética , Microscopía por Crioelectrón , ADN/química , ADN/genética , Conformación de Ácido Nucleico , ARN Guía de Kinetoplastida/genética
2.
Proc Natl Acad Sci U S A ; 121(37): e2404250121, 2024 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-39231203

RESUMEN

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.


Asunto(s)
Citomegalovirus , Proteínas del Envoltorio Viral , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Citomegalovirus/inmunología , Humanos , Animales , Ratones , Microscopía por Crioelectrón , Conformación Proteica , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Internalización del Virus , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Estabilidad Proteica , Vacunas contra Citomegalovirus/inmunología , Sustitución de Aminoácidos , Modelos Moleculares
3.
J Virol ; 97(10): e0092923, 2023 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-37737588

RESUMEN

IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.


Asunto(s)
Anticuerpos Antivirales , Epítopos de Linfocito B , Vacunas contra Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Vacunación , Proteínas Virales de Fusión , Humanos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/metabolismo
5.
Structure ; 30(9): 1331-1339.e3, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35738283

RESUMEN

The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KSQ, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KSQ and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KSQ and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KSQ active site, while an AT-swapped version of Pik127 demonstrated that KSQ can also decarboxylate malonyl groups. Mechanisms for how KSQ and KS domains catalyze carbon-carbon chemistry are proposed.


Asunto(s)
Carbono , Sintasas Poliquetidas , Aciltransferasas/química , Microscopía por Crioelectrón , Macrólidos , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética
6.
Nat Chem ; 13(10): 963-968, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34413500

RESUMEN

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded 'down' to an exposed 'up' state to bind the human angiotensin-converting enzyme 2 receptor and infect cells. While snapshots of the 'up' and 'down' states have been obtained by cryo-electron microscopy and cryo-electron tomagraphy, details of the RBD-opening transition evade experimental characterization. Here over 130 µs of weighted ensemble simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD-opening pathways. Together with ManifoldEM analysis of cryo-electron microscopy data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408 and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein represents a landmark study for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.


Asunto(s)
Polisacáridos/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Microscopía por Crioelectrón , Humanos , Simulación de Dinámica Molecular
7.
bioRxiv ; 2021 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-33619492

RESUMEN

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded "down" to an exposed "up" state in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 µs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

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