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Structural biology experiments benefit significantly from state-of-the-art synchrotron data collection. One can acquire macromolecular crystallography (MX) diffraction data on large-area photon-counting pixel-array detectors at framing rates exceeding 1000 frames per second, using 200â Gbps network connectivity, or higher when available. In extreme cases this represents a raw data throughput of about 25â GBâ s-1, which is nearly impossible to deliver at reasonable cost without compression. Our field has used lossless compression for decades to make such data collection manageable. Many MX beamlines are now fitted with DECTRIS Eiger detectors, all of which are delivered with optimized compression algorithms by default, and they perform well with current framing rates and typical diffraction data. However, better lossless compression algorithms have been developed and are now available to the research community. Here one of the latest and most promising lossless compression algorithms is investigated on a variety of diffraction data like those routinely acquired at state-of-the-art MX beamlines.
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A correction in the paper by Lazo et al. [(2021). J. Synchrotron Rad. 28, 1649-1661] is made.
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The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012â photonsâ s-1), micro-focus (7â µm × 5â µm), low-divergence (1â mrad × 0.35â mrad) energy-tunable (5-18â keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.
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Síncrotrons , Cristalografia por Raios X , Substâncias Macromoleculares/químicaRESUMO
Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277â K to 300â K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.
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Cristalografia por Raios X/métodos , Substâncias Macromoleculares/química , Robótica/métodos , Desenho de Equipamento , Espalhamento a Baixo Ângulo , Software , Síncrotrons , Raios XRESUMO
Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30â keV photon energy range and delivers a flux of 4.0â ×â 1012â photonsâ s-1 at 1â Å into a 1â µmâ ×â 1.5â µm to 10â µmâ ×â 10â µm (Vâ ×â H) variable focus, expected to reach 5â ×â 1012â photonsâ s-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.
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Síncrotrons , Cristalografia , Cristalografia por Raios X , Coleta de Dados , Substâncias Macromoleculares , ViscosidadeRESUMO
The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.
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Proteínas de Bactérias/química , Enterococcus faecalis/metabolismo , Enterococcus faecium/metabolismo , Proteínas de Ligação às Penicilinas/química , Isoformas de Proteínas/química , Resistência beta-Lactâmica , Acilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Domínio Catalítico , Cefalosporinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/isolamento & purificação , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/metabolismo , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Resistência beta-Lactâmica/genéticaRESUMO
Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).
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Cristalografia por Raios X , Modelos Moleculares , Proteínas/química , Cristalografia por Raios X/métodos , Fosfatidilinositol 3-Quinases/química , Conformação Proteica , Pirofosfatases/químicaRESUMO
The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1â µm beam size and photon flux of 3 × 1012 photonsâ s-1 at a photon energy of 12.66â keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100â nm raster-scanning precision at over 10 grid-linepairsâ s-1 frequency for fly scans of a 200â µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750â Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.
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Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating and inhibitory receptors that has been identified in bony fish. Here, we characterized the structure of an activating NITR that is expressed by a cytotoxic natural killer (NK)-like cell line and that specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by X-ray crystallography revealed that the V-type domains of NITRs form homodimers resembling rearranging antigen-binding receptor heterodimers. CDR1 elements of both subunits of NITR dimers form ligand-binding surfaces that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immunological responses.
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Linfócitos B/imunologia , Peixes-Gato/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/química , Receptores Imunológicos/imunologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Cristalização , Cristalografia por Raios X , Dimerização , Humanos , Células Matadoras Naturais/metabolismo , Família Multigênica , Receptores de Antígenos de Linfócitos B/química , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Peixe-Zebra/imunologiaRESUMO
The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by ld- and dd-transpeptidases that generate 3â3 and 4â3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3â3 cross-linked, and LdtMt2 is the dominant ld-transpeptidase. There are four additional sequence paralogs of LdtMt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, LdtMt5, and found it to be structurally and functionally distinct. The structures of apo-LdtMt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to LdtMt2, the LdtMt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of LdtMt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that LdtMt5 is not a functionally redundant ld-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.
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Parede Celular/fisiologia , Mycobacterium tuberculosis/enzimologia , Peptidil Transferases/metabolismo , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Concentração de Íons de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiologia , Peptidoglicano/metabolismo , Peptidil Transferases/química , Peptidil Transferases/genética , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Tumour metastasis is the primary cause of death of cancer patients. Development of new therapeutics preventing tumour metastasis is urgently needed. Migrastatin is a natural product secreted by Streptomyces, and synthesized migrastatin analogues such as macroketone are potent inhibitors of metastatic tumour cell migration, invasion and metastasis. Here we show that these migrastatin analogues target the actin-bundling protein fascin to inhibit its activity. X-ray crystal structural studies reveal that migrastatin analogues bind to one of the actin-binding sites on fascin. Our data demonstrate that actin cytoskeletal proteins such as fascin can be explored as new molecular targets for cancer treatment, in a similar manner to the microtubule protein tubulin.
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Proteínas de Transporte/antagonistas & inibidores , Macrolídeos/química , Macrolídeos/farmacologia , Proteínas dos Microfilamentos/antagonistas & inibidores , Metástase Neoplásica/prevenção & controle , Piperidonas/química , Piperidonas/farmacologia , Actinas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Macrolídeos/metabolismo , Macrolídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Mutação/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/patologia , Piperidonas/metabolismo , Piperidonas/uso terapêutico , Conformação ProteicaRESUMO
Phogrin/IA-2ß and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet ß-cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and ß-cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure-function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7.4 and 4.6 has been solved at 1.95- and 2.01-Å resolution, respectively. Similarly to the ME of ICA512, ME phogrin adopts a ferredoxin-like fold: a sheet of four antiparallel ß-strands packed against two α-helices. Sequence conservation among vertebrates, plants and insects suggests that the structural similarity extends to all the receptor family. Crystallized ME phogrin is monomeric, in agreement with solution studies but in striking contrast with the behavior of homodimeric ME ICA512. The structural details that may cause the quaternary structure differences are analyzed. The results provide a basis for building models of the overall orientation and oligomerization state of the receptor in biological membranes.
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Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Homologia de Sequência de Aminoácidos , Soluções , Relação Estrutura-AtividadeRESUMO
Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure of Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.
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Proteínas de Bactérias/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Proteínas de Bactérias/genética , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Staphylococcus aureus/genética , VirulênciaRESUMO
Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.
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Glicóis/química , Muramidase/química , Cristalografia por Raios X , Estrutura Terciária de ProteínaRESUMO
Idiosyncratic adverse drug reactions are unpredictable, dose-independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkages between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8(+) T cells that required HLA-B*57:01 molecules for their function; however, the mechanism by which abacavir induces this pathologic T-cell response remains unclear. Here we show that abacavir can bind within the F pocket of the peptide-binding groove of HLA-B*57:01, thereby altering its specificity. This provides an explanation for HLA-linked idiosyncratic adverse drug reactions, namely that drugs can alter the repertoire of self-peptides presented to T cells, thus causing the equivalent of an alloreactive T-cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir and that were recognized by T cells of hypersensitive patients. The assays that we have established can be applied to test additional compounds with suspected HLA-linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA-linked hypersensitivities, and guide the development of safer drugs.
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Hipersensibilidade a Drogas , Complexo Principal de Histocompatibilidade , Peptídeos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Modelos MolecularesRESUMO
Filopodia are cell surface protrusions that are essential for cell migration. This finger-like structure is supported by rigid tightly bundled actin filaments. The protein responsible for actin bundling in filopodia is fascin. However, the mechanism by which fascin functions in filopodial formation is not clear. Here we provide biochemical, cryo-electron tomographic, and x-ray crystal structural data demonstrating the unique structural characteristics of fascin. Systematic mutagenesis studies on 100 mutants of fascin indicate that there are two major actin-binding sites on fascin. Crystal structures of four fascin mutants reveal concerted conformational changes in fascin from inactive to active states in the process of actin bundling. Mutations in any one of the actin-binding sites impair the cellular function of fascin in filopodial formation. Altogether, our data reveal the molecular mechanism of fascin function in filopodial formation.
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Proteínas de Transporte/química , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/química , Pseudópodes/metabolismo , Actinas/química , Actinas/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência/métodos , Modelos Moleculares , Conformação Molecular , Metástase Neoplásica , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Transdução de SinaisRESUMO
Macromolecular crystallography contributes significantly to understanding diseases and, more importantly, how to treat them by providing atomic resolution 3D structures of proteins. This is achieved by collecting X-ray diffraction images of protein crystals from important biological pathways. Spotfinders are used to detect the presence of crystals with usable data, and the spots from such crystals are the primary data used to solve the relevant structures. Having fast and accurate spot finding is essential, but recent advances in synchrotron beamlines used to generate X-ray diffraction images have brought us to the limits of what the best existing spotfinders can do. This bottleneck must be removed so spotfinder software can keep pace with the X-ray beamline hardware improvements and be able to see the weak or diffuse spots required to solve the most challenging problems encountered when working with diffraction images. In this paper, we first present Bragg Spot Detection (BSD), a large benchmark Bragg spot image dataset that contains 304 images with more than 66 000 spots. We then discuss the open source extensible U-Net-based spotfinder Bragg Spot Finder (BSF), with image pre-processing, a U-Net segmentation backbone, and post-processing that includes artifact removal and watershed segmentation. Finally, we perform experiments on the BSD benchmark and obtain results that are (in terms of accuracy) comparable to or better than those obtained with two popular spotfinder software packages (Dozor and DIALS), demonstrating that this is an appropriate framework to support future extensions and improvements.
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SHP-1 (PTPN6) is a member of the SHP sub-family of protein tyrosine phosphatases and plays a critical role in the regulation of the JAK/STAT signaling pathway. Previous studies suggested that SHP-1 contains a PTP1B-like second phosphotyrosine pocket that allows for binding of tandem phosphotyrosine residues, such as those found in the activation loop of JAK kinases. To discover the structural nature of the interaction between SHP-1 and the JAK family member, JAK1, we determined the 1.8Å co-crystal structure of the SHP-1 catalytic domain and a JAK1-derived substrate peptide. This structure reveals electron density for only one bound phosphotyrosine residue. To investigate the role of the predicted second site pocket we determined the structures of SHP-1 in complex with phosphate and sulfate to 1.37Å and 1.7Å, respectively, and performed anomalous scattering experiments for a selenate-soaked crystal. These crystallographic data suggest that SHP-1 does not contain a PTP1B-like second site pocket. This conclusion is further supported by analysis of the relative dephosphorylation and binding affinities of mono- and tandem-phosphorylated peptide substrates. The crystal structures instead indicate that SHP-1 contains an extended C-terminal helix α2' incompatible with the predicted second phosphotyrosine binding site. This study suggests that SHP-1 defines a new category of PTP1B-like protein tyrosine phosphatases with a hindered second phosphotyrosine pocket.
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Janus Quinase 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Calorimetria , Domínio Catalítico , Janus Quinase 1/química , Ligação Proteica , Estrutura Secundária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Difração de Raios XRESUMO
Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5 Å crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication.
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Acetiltransferases/metabolismo , Phycodnaviridae/enzimologia , Poliaminas/metabolismo , Proteínas Virais/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Cristalografia por Raios X , Histonas/metabolismo , Cinética , Phycodnaviridae/química , Phycodnaviridae/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a D-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein-peptide-antibiotic complex. The 2.05 Å resolution MBP-peptide-teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.