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D-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-L-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography-small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products.
Assuntos
Produtos Biológicos , S-Adenosilmetionina , Aminoácidos , Antibacterianos , PeptídeosRESUMO
X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal's domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.
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Piscadela , Proteínas , Difração de Raios X , Raios X , Espalhamento a Baixo Ângulo , Proteínas/química , Cristalografia por Raios XRESUMO
Cyclic-olefin-copolymer (COC)-based microfluidic devices are increasingly becoming the center of highly valuable research for in situ X-ray measurements due to their compatibility with X-rays, biological compounds, chemical resistance, optical properties, low cost, and simplified handling. COC microfluidic devices present potential solutions to challenging biological applications such as protein binding, folding, nucleation, growth kinetics, and structural changes. In recent years, the techniques applied to manufacturing and handling these devices have capitalized on enormous progress toward small-scale sample probing. Here, we describe the new and innovative design aspects, fabrication, and experimental implementation of low-cost and micron-sized X-ray-compatible microfluidic sample environments that address diffusion-based crystal formation for crystallographic characterization. The devices appear fully compatible with crystal growth and subsequent X-ray diffraction experiments, resulting in remarkably low background data recording. The results highlighted in this research demonstrate how the engineered microfluidic devices allow the recording of accurate crystallographic data at room temperature and structure determination at high resolution.
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An approach for serial crystallography experiments based on wedged-data collection is described. This is an alternative method for recording in situ X-ray diffraction data on crystalline samples efficiently loaded in an X-ray compatible microfluidic chip. Proper handling of the microfluidic chip places crystalline samples at geometrically known positions with respect to the focused X-ray interaction area for serial data collection of small wedges. The integration of this strategy takes advantage of the greatly modular sample environment available on the endstation, which allows access to both in situ and more classical cryo-crystallography with minimum time loss. The method represents another optional data collection approach that adds up to the already large set of methods made available to users. Coupled with the advances in processing serial crystallography data, the wedged-data collection strategy proves highly efficient in minimizing the amount of required sample crystals for recording a complete dataset. From the advances in microfluidic technology presented here, high-throughput room-temperature crystallography experiments may become routine and should be easily extended to industrial use.
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Cristalografia por Raios X , Coleta de Dados , Difração de Raios XRESUMO
By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3-6, such as a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-L-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme-substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry.
Assuntos
Proteínas Arqueais , Methanosarcina , S-Adenosilmetionina , Proteínas Arqueais/química , Espectroscopia de Ressonância de Spin Eletrônica , Methanosarcina/enzimologia , Metilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/química , Vitamina B 12RESUMO
BACKGROUND: The Pacific Beetle Cockroach is the only known viviparous cockroach. The pregnant females provide nutrition to the embryos by secreting milk proteins (Lili-Mips), which crystallize in vivo. The crystals that grow in the embryo are heterogeneous in their protein sequence. It is not apparent from the structure determined what role heterogeneity and glycosylation played in crystallization. Lili-Mips are very nutritious. METHODS: Here, we report the cloning of synthesized Lili-Mip genes, their expression in Saccharomyces cerevisiae as secreted proteins, purification, crystallization, and the determination of a three-dimensional structure of one glycosylated and one deglycosylated form. RESULTS: A 2.35 Å structure of the glycosylated form is bound to palmitoleic acid and has several Zn atom mediated interactions. A 1.45 Å structure of the deglycosylated protein reveals a binding pocket that has both oleic and palmitoleic acid bound. Mass-spectrometry shows that oleic acid and palmitoleic acid are bound to the protein. Docking studies suggest that aliphatic chains of lengths 15, 16, and 18 carbons bind well in the pocket. CONCLUSIONS: The recombinantly expressed and secreted protein is glycosylated, has a bound fatty acid, is homogenous in its protein sequences, and readily forms crystals. The deglycosylated protein also crystallizes readily, suggesting that the high crystallizability of this protein is independent of glycosylation. GENERAL SIGNIFICANCE: Lili-Mips belong to the ubiquitous lipocalin family of proteins that bind to a large variety of ligands. While the residues lining the barrel are essential for the affinity of the ligand, our results show the role of side-chain orientations to ligand selectivity.
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Proteínas de InsetosRESUMO
Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red lightabsorbing) and Pfr (far-red lightabsorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/ß-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.
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The undulator beamline PROXIMA-1 at Synchrotron SOLEIL scheduled its first users in March 2008. The endstation is dedicated to biomolecular crystallography experiments, with a layout designed to favour anomalous data recording and studies of crystals with large cell dimensions. In 12 years, the beamline has accommodated 4267 shifts of 8â h and more than 6300 visitors. By the end of 2020, it saw 1039 identified published scientific papers referring to 1415 coordinates deposited in the Protein Data Bank. The current paper describes the PROXIMA-1 beamline, including the recent specific implementations developed for the sample environment. The setup installed in the experimental station contains numerous beam-shaping equipment, a chi-geometry three-axis goniometer, a single-photon-counting pixel-array X-ray detector, combined with a medium-throughput sample exchange robot. As part of a standard experimental scheme, PROXIMA-1 can also be accessed via `mail-in' services or remotely.
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A microfluidic laboratory recently opened at Synchrotron SOLEIL, dedicated to in-house research and external users. Its purpose is to provide the equipment and expertise that allow the development of microfluidic systems adapted to the beamlines of SOLEIL as well as other light sources. Such systems can be used to continuously deliver a liquid sample under a photon beam, keep a solid sample in a liquid environment or provide a means to track a chemical reaction in a time-resolved manner. The laboratory provides all the amenities required for the design and preparation of soft-lithography microfluidic chips compatible with synchrotron-based experiments. Three examples of microfluidic systems that were used on SOLEIL beamlines are presented, which allow the use of X-ray techniques to study physical, chemical or biological phenomena.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.
Assuntos
Elétrons , Lasers , Microtúbulos/ultraestrutura , Imagem Molecular/métodos , Tubulina (Proteína)/ultraestrutura , Algoritmos , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Processamento de Imagem Assistida por Computador , Imagem Molecular/instrumentação , Espalhamento de Radiação , Síncrotrons , Raios XRESUMO
MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF - the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.
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In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.
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Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.
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Movimento (Física) , Proteínas/química , Difração de Raios X/métodos , Acetilcolina/química , Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodosRESUMO
The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce background scattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude.
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A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.
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Microfluídica/métodos , Difração de Raios X/métodos , Cristalização/instrumentação , Cristalização/métodos , Microfluídica/instrumentação , Difração de Raios X/instrumentaçãoRESUMO
Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid's C-4 substituent's size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.
Assuntos
Antivirais/química , Borboletas , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteína HN/química , Respirovirus/efeitos dos fármacos , Animais , Antivirais/farmacologia , Sítios de Ligação , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteína HN/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Respirovirus/enzimologiaRESUMO
A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked ß-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids.
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Actinas/química , Amiloide/química , Escherichia coli/química , Fímbrias Bacterianas/química , Lasers , Raios X , Amiloide/ultraestrutura , Fímbrias Bacterianas/ultraestruturaRESUMO
Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.
Assuntos
Antimaláricos/farmacologia , Indóis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Quinona Redutases/metabolismo , Animais , Antimaláricos/química , Células CHO , Cricetulus , Radicais Livres/metabolismo , Humanos , Indóis/química , Modelos Moleculares , Estrutura Molecular , Plasmodium falciparum/metabolismo , Ligação Proteica , Conformação Proteica , Quinona Redutases/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.