RESUMO
In nitrogenase biosynthesis, the iron-molybdenum cofactor (FeMo-co) is externally assembled at scaffold proteins and delivered to the NifDK nitrogenase component by the NafY metallochaperone. Here we have used nuclear magnetic resonance, molecular dynamics, and functional analysis to elucidate the environment and coordination of FeMo-co in NafY. H121 stands as the key FeMo-co ligand. Regions near FeMo-co diverge from H121 and include the η1, α1, α2 helical lobe and a narrow path between H121 and C196.
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The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in 1 H MRI are impaired by the high abundance of protons in the body. 129 Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional 1 H MRI because of its ability to report specific chemical environments. To date, the scope of responsive 129 Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane-based 129 Xe hyperCEST NMR contrast agent that can be turned on in response to H2 O2 , which is upregulated in several disease states. Added H2 O2 was detected by 129 Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H2 O2 produced by HEKâ 293T cells activated with tumor necrosis factor.
Assuntos
Peróxido de Hidrogênio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Rotaxanos/uso terapêuticoRESUMO
Advancement of hyperpolarized 129 Xe MRI technology toward clinical settings demonstrates the considerable interest in this modality for diagnostic imaging. The number of contrast agents, termed biosensors, for 129 Xe MRI that respond to specific biological targets, has grown and diversified. Directly functionalized xenon-carrying macrocycles, such as the large family of cryptophane-based biosensors, are good for localization-based imaging and provide contrast before and after binding events occur. Noncovalently functionalized constructs, such as cucurbituril- and cyclodextrin-based biosensors, benefit from commercial availability and optimal exchange dynamics for CEST imaging. In this work, we report the first directly functionalized cucurbituril used as a xenon biosensor. Biotinylated cucurbit[7]uril (btCB7) gives rise to a 129 Xe hyperCEST response at the unusual shift of δ=28â ppm when bound to its protein target with substantial CEST contrast. We posit that the observed chemical shift is due to the deformation of btCB7 upon binding to avidin, caused by proximity to the protein surface. Conformational searches and molecular dynamics (MD) simulations support this hypothesis. This construct combines the strengths of both families of biosensors, enables a multitude of biological targets through avidin conjugation, and demonstrates the advantages of functionalized cucurbituril-based biosensors.
Assuntos
Avidina/química , Técnicas Biossensoriais/métodos , Biotina/química , Hidrocarbonetos Aromáticos com Pontes/química , Imidazóis/química , Ciclodextrinas/química , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Simulação de Dinâmica Molecular , Compostos Policíclicos/química , Ligação Proteica , Isótopos de XenônioRESUMO
Conformational control in peptoids, N-substituted glycines, is crucial for the design and synthesis of biologically-active compounds and atomically-defined nanomaterials. While there are a growing number of structural studies in solution, most have been performed with conformationally-constrained short sequences (e.g., sterically-hindered sidechains or macrocyclization). Thus, the inherent degree of heterogeneity of unconstrained peptoids in solution remains largely unstudied. Here, we explored the folding landscape of a series of simple peptoid tetramers in aqueous solution by NMR spectroscopy. By incorporating specific 13 C-probes into the backbone using bromoacetic acid-2-13 C as a submonomer, we developed a new technique for sequential backbone assignment of peptoids based on the 1,n-Adequate pulse sequence. Unexpectedly, two of the tetramers, containing an N-(2-aminoethyl)glycine residue (Nae), had preferred conformations. NMR and molecular dynamics studies on one of the tetramers showed that the preferred conformer (52%) had a trans-cis-trans configuration about the three amide bonds. Moreover, >80% of the ensemble contained a cis amide bond at the central amide. The backbone dihedral angles observed fall directly within the expected minima in the peptoid Ramachandran plot. Analysis of this compound against similar peptoid analogs suggests that the commonly used Nae monomer plays a key role in the stabilization of peptoid structure via a side-chain-to-main-chain interaction. This discovery may offer a simple, synthetically high-yielding approach to control peptoid structure, and suggests that peptoids have strong intrinsic conformational preferences in solution. These findings should facilitate the predictive design of folded peptoid structures, and accelerate application in areas ranging from drug discovery to biomimetic nanoscience.
Assuntos
Peptoides/química , Água/química , Isótopos de Carbono/química , Isomerismo , Simulação de Dinâmica Molecular , Nanoestruturas/química , Ressonância Magnética Nuclear Biomolecular , Peptoides/síntese química , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Teoria QuânticaRESUMO
Women and some racial and ethnic groups remain underrepresented in chemistry departments across the United States, and generally, efforts to improve representation have resulted in minimal or no improvements in the last 10 years. Here, we present the outcomes of a graduate-student-led initiative that sought to assess the issues affecting inclusivity, diversity, and wellness within the Department of Chemistry at the University of California, Berkeley. We report how the results of a department-tailored academic climate survey were used to develop a method to foster open, productive discussion among graduate students, postdoctoral researchers, and faculty. This event format led to an improved understanding of the challenges facing our community members, as well as the identification of strategies that can be used to make the Department of Chemistry more welcoming for all members. We report the success of this student-led effort to highlight the value of assessing diversity and inclusion at the department-level, as well as the benefits of using community data to stimulate productive, evidence-based discussions. Furthermore, we envision that these methods can be implemented within any research-focused academic community to promote positive cultural change.
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We report a CB6 rotaxane for the 129Xe hyperCEST NMR detection of matrix metalloprotease 2 (MMP-2) activity. MMP-2 is overexpressed in cancer tissue, and hence is a cancer marker. A peptide containing an MMP-2 recognition sequence was incorporated into the rotaxane, synthesized via CB6-promoted click chemistry. Upon cleavage of the rotaxane by MMP-2, CB6 became accessible for 129Xe@CB6 interactions, leading to protease-responsive hyperCEST activation.
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Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA+ ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. We identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1-σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.
Assuntos
Bactérias/química , Bactérias/enzimologia , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/metabolismo , Transcrição Gênica , Trifosfato de Adenosina/metabolismo , Bactérias/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Polimerase Sigma 54/genéticaRESUMO
Structural characterization of amyloid rich in cross-ß structures is crucial for unraveling the molecular basis of protein misfolding and amyloid formation associated with a wide range of human disorders. Elucidation of the ß-sheet structure in noncrystalline amyloid has, however, remained an enormous challenge. Here we report structural analyses of the ß-sheet structure in a full-length transthyretin amyloid using solid-state NMR spectroscopy. Magic-angle-spinning (MAS) solid-state NMR was employed to investigate native-like ß-sheet structures in the amyloid state using selective labeling schemes for more efficient solid-state NMR studies. Analyses of extensive long-range (13)C-(13)C correlation MAS spectra obtained with selectively (13)CO- and (13)Cα-labeled TTR reveal that the two main ß-structures in the native state, the CBEF and DAGH ß-sheets, remain intact after amyloid formation. The tertiary structural information would be of great use for examining the quaternary structure of TTR amyloid.
Assuntos
Amiloide/química , Espectroscopia de Ressonância Magnética/métodos , Pré-Albumina/química , Dicroísmo Circular , Conformação ProteicaRESUMO
The traditional structure-function paradigm has provided significant insights for well-folded proteins in which structures can be easily and rapidly revealed by X-ray crystallography beamlines. However, approximately one-third of the human proteome is comprised of intrinsically disordered proteins and regions (IDPs/IDRs) that do not adopt a dominant well-folded structure, and therefore remain "unseen" by traditional structural biology methods. This Perspective considers the challenges raised by the "Dark Proteome", in which determining the diverse conformational substates of IDPs in their free states, in encounter complexes of bound states, and in complexes retaining significant disorder requires an unprecedented level of integration of multiple and complementary solution-based experiments that are analyzed with state-of-the art molecular simulation, Bayesian probabilistic models, and high-throughput computation. We envision how these diverse experimental and computational tools can work together through formation of a "computational beamline" that will allow key functional features to be identified in IDP structural ensembles.
Assuntos
Biologia Computacional , Proteínas Intrinsicamente Desordenadas/química , Proteoma , Teorema de Bayes , Cromatografia em Gel , Cristalografia por Raios X , Genoma Humano , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Probabilidade , Conformação Proteica , Dobramento de Proteína , Proteômica/métodos , SoftwareRESUMO
We have synthesized targeted, selective, and highly sensitive (129)Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized (129)Xe NMR signal contrast and hyper-CEST (129)Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized (129)Xe NMR nanoscale biosensor for future applications in cancer detection in vivo.
Assuntos
Técnicas Biossensoriais/métodos , Imagem Molecular/métodos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Linhagem Celular Tumoral , Humanos , Levivirus , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Compostos Policíclicos/química , Conformação ProteicaRESUMO
We report a (129)Xe NMR relaxation-based sensing approach that exploits changes in the bulk xenon relaxation rate induced by slowed tumbling of a cryptophane-based sensor upon target binding. The amplification afforded by detection of the bulk dissolved xenon allows sensitive detection of targets. The sensor comprises a xenon-binding cryptophane cage, a target interaction element, and a metal chelating agent. Xenon associated with the target-bound cryptophane cage is rapidly relaxed and then detected after exchange with the bulk. Here we show that large macromolecular targets increase the rotational correlation time of xenon, increasing its relaxation rate. Upon binding of a biotin-containing sensor to avidin at 1.5 µM concentration, the free xenon T2 is reduced by a factor of 4.
Assuntos
Técnicas Biossensoriais , Substâncias Macromoleculares/química , Isótopos de Xenônio/química , Biotina/química , Quelantes/química , Espectroscopia de Ressonância Magnética , Metais/química , Peso Molecular , Peptídeos/química , Compostos Policíclicos , Ligação Proteica , Solubilidade , Água/químicaRESUMO
Studies of hyperpolarized xenon-129 (hp-(129)Xe) in media such as liquid crystals and cell suspensions are in demand for applications ranging from biomedical imaging to materials engineering but have been hindered by the inability to bubble Xe through the desired media as a result of viscosity or perturbations caused by bubbles. Herein a device is reported that can be reliably used to dissolve hp-(129)Xe into viscous aqueous and organic samples without bubbling. This method is robust, requires small sample volumes (<60â µL), is compatible with existing NMR hardware, and is made from readily available materials. Experiments show that Xe can be introduced into viscous and aligned media without disrupting molecular order. We detected dissolved xenon in an aqueous liquid crystal that is disrupted by the shear forces of bubbling, and we observed liquid-crystal phase transitions in (MBBA). This tool allows an entirely new class of samples to be investigated by hyperpolarized-gas NMR spectroscopy.
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Elucidation of structural changes involved in protein misfolding and amyloid formation is crucial for unraveling the molecular basis of amyloid formation. Here we report structural analyses of the amyloidogenic intermediate and amyloid aggregates of transthyretin using solution and solid-state nuclear magnetic resonance (NMR) spectroscopy. Our solution NMR results show that one of the two main ß-sheet structures (CBEF ß-sheet) is maintained in the aggregation-competent intermediate, while the other DAGH ß-sheet is more flexible on millisecond time scales. Magic-angle-spinning solid-state NMR revealed that AB loop regions interacting with strand A in the DAGH ß-sheet undergo conformational changes, leading to the destabilized DAGH ß-sheet.
Assuntos
Amiloide/química , Modelos Moleculares , Pré-Albumina/química , Agregação Patológica de Proteínas/patologia , Substituição de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Amiloide/ultraestrutura , Dimerização , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Mutação , Ressonância Magnética Nuclear Biomolecular , Pré-Albumina/genética , Pré-Albumina/metabolismo , Agregação Patológica de Proteínas/etiologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Redobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes , SolubilidadeRESUMO
In order to understand factors controlling the synthesis and deposition of cellulose, we have studied the Arabidopsis (Arabidopsis thaliana) double mutant shaven3 shaven3-like1 (shv3svl1), which was shown previously to exhibit a marked cellulose deficiency. We discovered that exogenous sucrose (Suc) in growth medium greatly enhances the reduction in hypocotyl elongation and cellulose content of shv3svl1 This effect was specific to Suc and was not observed with other sugars or osmoticum. Live-cell imaging of fluorescently labeled cellulose synthase complexes revealed a slowing of cellulose synthase complexes in shv3svl1 compared with the wild type that is enhanced in a Suc-conditional manner. Solid-state nuclear magnetic resonance confirmed a cellulose deficiency of shv3svl1 but indicated that cellulose crystallinity was unaffected in the mutant. A genetic suppressor screen identified mutants of the plasma membrane Suc/H(+) symporter SUC1, indicating that the accumulation of Suc underlies the Suc-dependent enhancement of shv3svl1 phenotypes. While other cellulose-deficient mutants were not specifically sensitive to exogenous Suc, the feronia (fer) receptor kinase mutant partially phenocopied shv3svl1 and exhibited a similar Suc-conditional cellulose defect. We demonstrate that shv3svl1, like fer, exhibits a hyperpolarized plasma membrane H(+) gradient that likely underlies the enhanced accumulation of Suc via Suc/H(+) symporters. Enhanced intracellular Suc abundance appears to favor the partitioning of carbon to starch rather than cellulose in both mutants. We conclude that SHV3-like proteins may be involved in signaling during cell expansion that coordinates proton pumping and cellulose synthesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Celulose/metabolismo , Sacarose/metabolismo , Simportadores/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Radioisótopos de Carbono/metabolismo , Parede Celular/metabolismo , Celulose/química , Mapeamento Cromossômico , Escuridão , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Concentração de Íons de Hidrogênio , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Hipocótilo/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fenótipo , Fosfotransferases , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Amido/química , Amido/metabolismo , Simportadores/genéticaRESUMO
The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.
Assuntos
Heptoses/metabolismo , Engenharia Metabólica/métodos , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Ativação Transcricional/fisiologia , Xilose/metabolismo , Ativação Enzimática , Inativação Gênica , Melhoramento Genético/métodos , Heptoses/genéticaRESUMO
We report a method for blocking interactions between (129)Xe and cucurbit[6]uril (CB6) until activation by a specific chemical event. We synthesized a CB6-rotaxane that allowed no (129)Xe interaction with the CB6 macrocycle component until a cleavage event released the CB6, which then produced a (129)Xe@CB6 NMR signal. This contrast-upon-activation (129)Xe NMR platform allows for modular synthesis and can be expanded to applications in detection and disease imaging.
RESUMO
We performed a screen for genetic suppressors of cobra, an Arabidopsis mutant with defects in cellulose formation and an increased ratio of unesterified/esterified pectin. We identified a suppressor named mongoose1 (mon1) that suppressed the growth defects of cobra, partially restored cellulose levels, and restored the esterification ratio of pectin to wild-type levels. mon1 was mapped to the MEDIATOR16 (MED16) locus, a tail mediator subunit, also known as SENSITIVE TO FREEZING6 (SFR6). When separated from the cobra mutation, mutations in MED16 caused resistance to cellulose biosynthesis inhibitors, consistent with their ability to suppress the cobra cellulose deficiency. Transcriptome analysis revealed that a number of cell wall genes are misregulated in med16 mutants. Two of these genes encode pectin methylesterase inhibitors, which, when ectopically expressed, partially suppressed the cobra phenotype. This suggests that cellulose biosynthesis can be affected by the esterification levels of pectin, possibly through modifying cell wall integrity or the interaction of pectin and cellulose.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Glicoproteínas de Membrana/genética , Mutação , Transativadores/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Celulose/análise , Celulose/biossíntese , Esterificação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glicoproteínas de Membrana/metabolismo , Monossacarídeos/análise , Monossacarídeos/metabolismo , Pectinas/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismoRESUMO
Recent work has shown that xenon chemical shifts in cryptophane-cage sensors are affected when tethered chelators bind to metals. Here, we explore the xenon shifts in response to a wide range of metal ions binding to diastereomeric forms of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) linked to cryptophane-A. The shifts induced by the binding of Ca(2+) , Cu(2+) , Ce(3+) , Zn(2+) , Cd(2+) , Ni(2+) , Co(2+) , Cr(2+) , Fe(3+) , and Hg(2+) are distinct. In addition, the different responses of the diastereomers for the same metal ion indicate that shifts are affected by partial folding with a correlation between the expected coordination number of the metal in the DOTA complex and the chemical shift of (129) Xe. These sensors may be used to detect and quantify many important metal ions, and a better understanding of the basis for the induced shifts could enhance future designs.
Assuntos
Quelantes/química , Compostos Heterocíclicos com 1 Anel/química , Metais Alcalinoterrosos/química , Metais Pesados/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Isótopos de Xenônio/químicaRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized ß-amyloid peptides with Aß42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aß42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aß42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aß42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aß42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aß42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aß42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aß42 peptide (Garai et al., 2009). We obtain a final yield of â¼ 6 mg/L culture for (15)N isotope-labeled Aß42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aß42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aß42 peptide as well as its other variants including any Aß42 peptide mutants.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Escherichia coli/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos beta-Amiloides/isolamento & purificação , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Humanos , Marcação por Isótopo , Isótopos de Nitrogênio/análise , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Viral protein genome-linked (VPg) proteins play a critical role in the life cycle of vertebrate and plant positive-sense RNA viruses by acting as a protein primer for genome replication and as a protein cap for translation initiation. Here we report the solution structure of the porcine sapovirus VPg core (VPg(C)) determined by multi-dimensional NMR spectroscopy. The structure of VPg(C) is composed of three α-helices stabilized by several conserved hydrophobic residues that form a helical bundle core similar to that of feline calicivirus VPg. The putative nucleotide acceptor Tyr956 within the first helix of the core is completely exposed to solvent accessible surface to facilitate nucleotidylation by viral RNA polymerase. Comparison of VPg structures suggests that the surface for nucleotidylation site is highly conserved among the Caliciviridae family, whereas the backbone core structures are different. These structural features suggest that caliciviruses share common mechanisms of VPg-dependent viral replication and translation.