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1.
Acta Crystallogr D Struct Biol ; 80(Pt 3): 194-202, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38411550

RESUMO

The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.


Assuntos
Anidrase Carbônica II , Anidrase Carbônica II/química , Cristalografia por Raios X , Proteínas/química , Síncrotrons , Raios X , Humanos
2.
Nat Commun ; 15(1): 389, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38195598

RESUMO

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.


Assuntos
Benchmarking , Sistemas Computacionais , Microscopia Crioeletrônica , Anisotropia , Coleta de Dados
3.
Nat Protoc ; 18(11): 3413-3459, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37735235

RESUMO

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.


Assuntos
Capsídeo , Dependovirus , Animais , Humanos , Capsídeo/química , Dependovirus/genética , Terapia Genética/métodos , Técnicas de Transferência de Genes , Proteínas do Capsídeo/genética , Vetores Genéticos , Transdução Genética
4.
bioRxiv ; 2023 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-37503021

RESUMO

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

5.
Viruses ; 14(9)2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-36146728

RESUMO

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25-45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to "switch on/off" the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.


Assuntos
Parvovirus B19 Humano , Internalização do Vírus , Proteínas do Capsídeo/metabolismo , Criança , Endossomos/metabolismo , Humanos , Parvovirus B19 Humano/metabolismo , Fosfolipases A2/química , Proteínas Virais/metabolismo
6.
J Virol ; 96(11): e0033522, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35532224

RESUMO

Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in the general population capable of neutralizing AAV vectors. Hence, there is a need for AAV vectors that can evade the preexisting immune response. One possible source of human naive vectors are AAVs that do not disseminate in the primate population, and one such example is serpentine AAV (SAAV). This study characterizes the structural and biophysical properties of the SAAV capsid and its receptor interactions and antigenicity. Single particle cryo-electron microscopy (cryo-EM) and thermal stability studies were conducted to characterize the SAAV capsid structure at pH 7.4, 6.0, 5.5, and 4.0, conditions experienced during cellular trafficking. Cell binding assays using Chinese hamster ovary (CHO) cell lines identified terminal sialic acid as the primary attachment receptor for SAAV similar to AAV1, 4, 5, and 6. The binding site of sialic acid to the SAAV capsid was mapped near the 2-fold axis toward the 2/5-fold wall, in a different location than AAV1, 4, 5, and 6. Towards determining the SAAV capsid antigenicity native immunodot blots showed that SAAV evades AAV serotype-specific mouse monoclonal antibodies. However, despite its reptilian origin, it was recognized by ~25% of 50 human sera tested, likely due to the presence of cross-reactive antibodies. These findings will inform future gene delivery applications using SAAV-based vectors and further aid the structural characterization and annotation of the repertoire of available AAV capsids. IMPORTANCE AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. In order to circumvent preexisting neutralizing antibodies targeting mammalian AAV capsids, serpentine AAV (SAAV) was evaluated as a potential alternative to existing mammalian therapeutic vectors. The SAAV capsid was found to be thermostable at a wide range of environmental pH conditions, and its structure showed conservation of the core capsid topology but displays high structural variability on the surface. At the same time, it binds to a common receptor, sialic acid, that is also utilized by other AAVs already being utilized in gene therapy trials. Contrary to the initial hypothesis, SAAV capsids were recognized by one in four human sera tested, pointing to conserved amino acids around the 5-fold region as epitopes for cross-reacting antibodies.


Assuntos
Capsídeo , Dependovirus , Animais , Células CHO , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Cricetinae , Cricetulus , Reações Cruzadas , Microscopia Crioeletrônica , Dependovirus/fisiologia , Epitopos , Vetores Genéticos , Humanos , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo
7.
Virology ; 565: 22-28, 2022 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-34638006

RESUMO

Adeno-associated virus (AAV) are classified as non-enveloped ssDNA viruses. However, AAV capsids embedded within exosomes have been observed, and it has been suggested that the AAV membrane associated accessory protein (MAAP) may play a role in envelope-associated AAV (EA-AAV) capsid formation. Here, we observed and selected sufficient homogeneous EA-AAV capsids of AAV2, produced using the Sf9 baculoviral expression system, to determine the cryo-electron microscopy (cryo-EM) structure at 3.14 Å resolution. The reconstructed map confirmed that the EA-AAV capsid, showed no significant structural variation compared to the non-envelope capsid. In addition, the Sf9 expression system used implies the notion that MAAP may enhance exosome AAV encapsulation. Furthermore, we speculate that these EA-AAV capsids may have therapeutic benefits over the currently used non-envelope AAV capsids, with advantages in immune evasion and/or improved infectivity.


Assuntos
Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Dependovirus/ultraestrutura , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Dependovirus/química , Exossomos , Evasão da Resposta Imune , Conformação Proteica , Células Sf9
8.
Viruses ; 12(6)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32560452

RESUMO

Several members of the Protoparvovirus genus, capable of infecting humans, have been recently discovered, including cutavirus (CuV) and tusavirus (TuV). To begin the characterization of these viruses, we have used cryo-electron microscopy and image reconstruction to determine their capsid structures to ~2.9 Å resolution, and glycan array and cell-based assays to identify glycans utilized for cellular entry. Structural comparisons show that the CuV and TuV capsids share common features with other parvoviruses, including an eight-stranded anti-parallel ß-barrel, depressions at the icosahedral 2-fold and surrounding the 5-fold axes, and a channel at the 5-fold axes. However, the viruses exhibit significant topological differences in their viral protein surface loops. These result in three separated 3-fold protrusions, similar to the bufaviruses also infecting humans, suggesting a host-driven structure evolution. The surface loops contain residues involved in receptor binding, cellular trafficking, and antigenic reactivity in other parvoviruses. In addition, terminal sialic acid was identified as the glycan potentially utilized by both CuV and TuV for cellular entry, with TuV showing additional recognition of poly-sialic acid and sialylated Lewis X (sLeXLeXLeX) motifs reported to be upregulated in neurotropic and cancer cells, respectively. These structures provide a platform for annotating the cellular interactions of these human pathogens.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/ultraestrutura , Parvovirus/fisiologia , Receptores Virais/metabolismo , Ligação Viral , Adulto , Sequência de Aminoácidos , Animais , Criança , Microscopia Crioeletrônica , Humanos , Ácido N-Acetilneuramínico/metabolismo , Infecções por Parvoviridae/patologia , Parvovirus/genética , Polissacarídeos/metabolismo , Conformação Proteica , Análise de Sequência de DNA
9.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31826994

RESUMO

Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB), thereby enabling the delivery of therapeutic genes to the central nervous system. Here, the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08-, 2.75-, and 3.39-Šresolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% identical to AAV8. However, the capsid structures of all three viruses are similar, with only minor differences observed in the previously described surface variable regions, suggesting that specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket, supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus The structural characterization of these viruses provides a platform for future vector engineering efforts toward improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity, or receptor retargeting.IMPORTANCE Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. The closely related AAVrh.39 has also shown promise in vivo As recently attained for other AAV biologics, e.g., Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain U.S. Food and Drug Administration approval for commercialization in the near future. This study characterized the capsid structures of these clinical vectors at atomic resolution using cryo-electron microscopy and image reconstruction for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for central nervous system delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/ultraestrutura , Capsídeo/química , Capsídeo/ultraestrutura , Dependovirus/química , Sequência de Aminoácidos , Barreira Hematoencefálica , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Dependovirus/genética , Terapia Genética , Vetores Genéticos , Células HEK293 , Humanos , Imageamento Tridimensional , Modelos Moleculares , Estados Unidos , United States Food and Drug Administration
10.
Viruses ; 11(5)2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035643

RESUMO

Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical.


Assuntos
Dependovirus/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/química , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/isolamento & purificação , Dependovirus/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/genética , Vírion/isolamento & purificação , Vírion/metabolismo , Vírion/ultraestrutura
11.
Nat Commun ; 9(1): 3628, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194371

RESUMO

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.


Assuntos
Capsídeo/ultraestrutura , Microscopia Crioeletrônica/métodos , Parvovirinae/ultraestrutura , Dependovirus , Rotavirus/ultraestrutura
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