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1.
J Biol Chem ; 300(6): 107410, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38796062

RESUMEN

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.


Asunto(s)
Citidina Desaminasa , ADN , Humanos , Desaminación , Citidina Desaminasa/metabolismo , ADN/metabolismo , ADN/química , Cinética , Desaminasas APOBEC/metabolismo , Inhibidores Enzimáticos/farmacología
2.
J Biol Chem ; 300(8): 107502, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38945452

RESUMEN

Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the in vivo applications of VHH-based biologics against OUD and overdose.


Asunto(s)
Fentanilo , Anticuerpos de Dominio Único , Fentanilo/química , Fentanilo/inmunología , Animales , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Humanos , Camelidae/inmunología , Camélidos del Nuevo Mundo , Analgésicos Opioides/química , Analgésicos Opioides/farmacología , Analgésicos Opioides/inmunología
3.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-35165203

RESUMEN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.


Asunto(s)
Exorribonucleasas/química , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Dominios Proteicos , ARN Viral/química , SARS-CoV-2/enzimología , Proteínas no Estructurales Virales/química , Sitios de Unión/genética , COVID-19/virología , Dominio Catalítico , Cristalografía por Rayos X , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Mutación Missense , Unión Proteica , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/fisiología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33658373

RESUMEN

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.


Asunto(s)
Proteínas Arqueales/química , ADN de Archaea/química , Endonucleasas/química , Pyrococcus furiosus/enzimología , Proteínas Arqueales/genética , Dominio Catalítico , ADN de Archaea/genética , Desaminación , Endonucleasas/genética , Pyrococcus furiosus/genética
5.
bioRxiv ; 2024 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-38766133

RESUMEN

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

6.
Viruses ; 14(8)2022 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-36016411

RESUMEN

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3'-to-5' exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3'-deoxy-3',4'-didehydro-cytidine 5'-monophosphate (ddhCMP), incorporated by RdRp at the 3' end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3'-deoxy-cytidine monophosphate (3'-dCMP), whereas RdRp is more susceptible to chain termination by 3'-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP.


Asunto(s)
COVID-19 , Exorribonucleasas , Antivirales/farmacología , Citidina/farmacología , Exorribonucleasas/metabolismo , Humanos , Mutación , Nucleótidos , ARN , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/genética , SARS-CoV-2 , Proteínas no Estructurales Virales/metabolismo , Replicación Viral
7.
bioRxiv ; 2021 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-33821277

RESUMEN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

8.
Viruses ; 13(4)2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33921405

RESUMEN

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Cristalización , Células HEK293 , Humanos , Inmunidad Innata , Simulación de Dinámica Molecular , Unión Proteica , Anticuerpos de Cadena Única/metabolismo
9.
Nat Commun ; 12(1): 7325, 2021 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-34916516

RESUMEN

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Cristalografía por Rayos X , Receptores de Antígenos/química , Receptores de Antígenos/inmunología , Tiburones/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Epítopos , Mutación , Filogenia , Unión Proteica , SARS-CoV-2 , Alineación de Secuencia , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus/inmunología
10.
Commun Biol ; 3(1): 691, 2020 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-33214665

RESUMEN

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages - a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.


Asunto(s)
Bacteriófago T4/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Virales/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Viral de la Expresión Génica , N-Acetil Muramoil-L-Alanina Amidasa/genética , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Proteínas Virales/química , Proteínas Virales/genética
11.
Acta Crystallogr D Struct Biol ; 76(Pt 9): 899-904, 2020 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-32876065

RESUMEN

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Šresolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.


Asunto(s)
Proteínas Virales/química , Conformación Proteica
12.
Nat Commun ; 11(1): 3121, 2020 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-32561747

RESUMEN

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Šresolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.


Asunto(s)
Interacciones Huésped-Patógeno/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Integrasas/metabolismo , Proteína Fosfatasa 2/genética , Proteínas Virales/metabolismo , Integración Viral/genética , Microscopía por Crioelectrón , ADN Viral/metabolismo , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/enzimología , Humanos , Integrasas/ultraestructura , Modelos Moleculares , Mutación Puntual , Unión Proteica/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/ultraestructura , Proteínas Virales/ultraestructura
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