RESUMO
ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas do Citoesqueleto/metabolismo , N-Glicosil Hidrolases/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP-Ribosilação , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Domínio Catalítico , Proteínas do Citoesqueleto/antagonistas & inibidores , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/imunologia , Escherichia coli/metabolismo , Humanos , Mutagênese Sítio-Dirigida , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Serratia/metabolismo , Imagem com Lapso de TempoRESUMO
The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a â¼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2-specific sequence, 73YIDI76 Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.
Assuntos
Estrutura Molecular , SARS-CoV-2/química , Proteínas Virais/química , Evolução Molecular , Evasão da Resposta ImuneRESUMO
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
Assuntos
Cristalografia por Raios X/métodos , Lasers , Acústica , Complexo de Proteína do Fotossistema II/química , Fitocromo/química , Ribonucleotídeo Redutases/química , Espectrometria por Raios X/métodosRESUMO
Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33â transfersâ s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.
Assuntos
Acústica , Ensaios de Triagem em Larga Escala/métodos , Proteínas/química , Sefarose/química , Cristalização , HidrogéisRESUMO
Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.
Assuntos
Acústica , Cristalização/métodos , Proteínas/química , Bibliotecas de Moléculas Pequenas , Acústica/instrumentação , Cristalização/instrumentação , Cristalografia por Raios X , Descoberta de Drogas , Desenho de Equipamento , Muramidase/química , Termolisina/química , Tripsina/químicaRESUMO
X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from â¼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20â µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.
Assuntos
Cristalografia por Raios X/métodos , Insulina/química , Insulina/efeitos da radiação , Muramidase/química , Muramidase/efeitos da radiação , Espalhamento a Baixo Ângulo , Humanos , Próteses e Implantes , Solventes/química , Síncrotrons , Difração de Raios XRESUMO
Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.
Assuntos
Cristalografia por Raios X/instrumentação , Lentes , Substâncias Macromoleculares/química , Síncrotrons/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , New York , Espalhamento de RadiaçãoRESUMO
Nutrient import across Gram-negative bacteria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4-ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB4-ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB4-ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6-ExbD4, and propose a structural arrangement of its transmembrane α-helical domains.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Polímeros/química , Propilaminas/química , Tensoativos/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Sítios de Ligação , Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , SolubilidadeRESUMO
Targeted alpha therapy (TAT) pairs the specificity of antigen targeting with the lethality of alpha particles to eradicate cancerous cells. Actinium-225 [225Ac; t1/2 = 9.920(3) days] is an alpha-emitting radioisotope driving the next generation of TAT radiopharmaceuticals. Despite promising clinical results, a fundamental understanding of Ac coordination chemistry lags behind the rest of the Periodic Table due to its limited availability, lack of stable isotopes, and inadequate systems poised to probe the chemical behavior of this radionuclide. In this work, we demonstrate a platform that combines an 8-coordinate synthetic ligand and a mammalian protein to characterize the solution and solid-state behavior of the longest-lived Ac isotope, 227Ac [t1/2 = 21.772(3) years]. We expect these results to direct renewed efforts for 225Ac-TAT development, aid in understanding Ac coordination behavior relative to other +3 lanthanides and actinides, and more broadly inform this element's position on the Periodic Table.
Assuntos
Actínio , Quelantes , Actínio/química , Quelantes/química , Cristalização , Compostos Radiofarmacêuticos/química , Humanos , LigantesRESUMO
Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.
Assuntos
Proteína gp41 do Envelope de HIV , HIV-1 , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Rhodopseudomonas palustris metabolizes aromatic compounds derived from lignin degradation products and has the potential for bioremediation of xenobiotic compounds. We recently identified four possible solute-binding proteins in R. palustris that demonstrated binding to aromatic lignin monomers. Characterization of these proteins in the absence and presence of the aromatic ligands will provide unprecedented insights into the specificity and mode of aromatic ligand binding in solute-binding proteins. Here, we report the thermodynamic and structural properties of the proteins with aromatic ligands using isothermal titration calorimetry, small/wide angle x-ray scattering, and theoretical predictions. The proteins exhibit high affinity for the aromatic substrates with dissociation constants in the low micromolar to nanomolar range. The global shapes of the proteins are characterized by flexible ellipsoid-like structures with maximum dimensions in the 80-90-Å range. The data demonstrate that the global shapes remained unaltered in the presence of the aromatic ligands. However, local structural changes were detected in the presence of some ligands, as judged by the observed features in the wide angle x-ray scattering regime at q ~0.20-0.40 Å(-1). The theoretical models confirmed the elongated nature of the proteins and showed that they consist of two domains linked by a hinge. Evaluation of the protein-binding sites showed that the ligands were found in the hinge region and that ligand stabilization was primarily driven by hydrophobic interactions. Taken together, this study shows the capability of identifying solute-binding proteins that interact with lignin degradation products using high throughput genomic and biophysical approaches, which can be extended to other organisms.
Assuntos
Proteínas de Bactérias/química , Hidrocarbonetos Aromáticos/química , Estrutura Terciária de Proteína , Termodinâmica , Algoritmos , Proteínas de Bactérias/metabolismo , Benzoatos/química , Benzoatos/metabolismo , Calorimetria , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Hidrocarbonetos Aromáticos/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Parabenos/química , Parabenos/metabolismo , Ligação Proteica , Rodopseudomonas/metabolismo , Espalhamento a Baixo Ângulo , Tirosina/química , Tirosina/metabolismo , Difração de Raios XRESUMO
To take full advantage of advanced data collection techniques and high beam flux at next-generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal-containing droplets onto a solid X-ray transparent surface, which can then be positioned and rotated for data collection. Proof-of-concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
Assuntos
Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Proteínas/química , Coleta de Dados , Substâncias Macromoleculares , SíncrotronsRESUMO
One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Bactérias/química , Proteínas de Ligação a DNA/química , RNA Polimerase Sigma 54/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Bactérias/genética , Bactérias/metabolismo , Berílio/química , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluoretos/química , Expressão Gênica , Estrutura Terciária de Proteína , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição GênicaRESUMO
BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an â¼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.
Assuntos
Antígenos CD/metabolismo , Evolução Molecular , Glicoproteínas de Membrana/metabolismo , Liberação de Vírus/fisiologia , Vírus/metabolismo , Animais , Antígenos CD/química , Antígenos CD/genética , Cristalografia por Raios X , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Two transmission-mode diamond X-ray beam position monitors installed at National Synchrotron Light Source (NSLS) beamline X25 are described. Each diamond beam position monitor is constructed around two horizontally tiled electronic-grade (p.p.b. nitrogen impurity) single-crystal (001) CVD synthetic diamonds. The position, angle and flux of the white X-ray beam can be monitored in real time with a position resolution of 500â nm in the horizontal direction and 100â nm in the vertical direction for a 3â mm × 1â mm beam. The first diamond beam position monitor has been in operation in the white beam for more than one year without any observable degradation in performance. The installation of a second, more compact, diamond beam position monitor followed about six months later, adding the ability to measure the angular trajectory of the photon beam.
Assuntos
Síncrotrons/instrumentação , Diamante/química , Desenho de Equipamento , Raios XRESUMO
Regulation of the major Ser/Thr phosphatase protein phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry, and small-angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and inhibitor 2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1-spinophilin-I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1-spinophilin and PP1-I-2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1-I-2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multifunctional PP1 holoenzymes.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas/metabolismo , Sítios de Ligação , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/fisiologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Ligação Proteica , Multimerização Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/fisiologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/fisiologia , Espalhamento de Radiação , Relação Estrutura-Atividade , Difração de Raios XRESUMO
We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 Ǻ resolution crystal structures.
Assuntos
Acústica , Cristalografia por Raios X/métodosRESUMO
In recent years there has been a growing interest in the application of X-ray scattering techniques to biomolecules in solution. At NSLS, a new undulator-based beamline, X9, has been constructed to address the oversubscribed user demand for X-ray scattering. Beamline X9 has the capability to perform small/wide-angle X-ray scattering (SAXS/WAXS) all in one single instrument. This is accomplished by utilizing a vacuum sample/detector chamber that is an integral part of the SAXS scattering flight path. This vacuum chamber allows a WAXS detector to be positioned at a close distance from the sample, while not interfering with scattered X-rays at small angles from reaching the SAXS detector. A regular training program, the X9 workbench, has also been established to allow users to become familiar with beamline X9 for solution X-ray scattering.
Assuntos
Espalhamento a Baixo Ângulo , Síncrotrons/instrumentação , Difração de Raios X/métodos , Cristalografia por Raios X/métodos , DNA/química , RNA/química , Espalhamento de Radiação , Raios XRESUMO
Amelogenin undergoes self-assembly and plays an essential role in guiding enamel mineral formation. The leucine-rich amelogenin peptide (LRAP) is an alternative splice product of the amelogenin gene and is composed of the N terminus (containing the only phosphate group) and the C terminus of full-length amelogenin. This study was conducted to investigate further the role of phosphorylation in LRAP self-assembly in the presence and absence of calcium using small angle X-ray scattering (SAXS). Consistent with our previous dynamic light-scattering findings for phosphorylated (+P) and non-phosphorylated (-P) LRAP, SAXS analyses revealed radii of gyration (R(g)) for LRAP(-P) (46.3-48.0 Å) that were larger than those for LRAP(+P) (25.0-27.4 Å) at pH 7.4. However, added calcium (up to 2.5 mM) induced significant increases in the R(g) of LRAP(+P) (up to 46.4 Å), while it had relatively little effect on LRAP(-P) particle size. Furthermore, SAXS analyses suggested compact folded structures for LRAP(-P) in the presence and absence of calcium, whereas the conformation of LRAP(+P) changed from an unfolded structure to a more compact structure upon the addition of calcium. We conclude that the single phosphate group in LRAP(+P) induces functionally important conformational changes, suggesting that phosphorylation may also influence amelogenin conformation and protein-mineral interactions during the early stages of amelogenesis.
Assuntos
Amelogenina/química , Calcificação Fisiológica , Cálcio/química , Proteínas do Esmalte Dentário/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Nanopartículas , Tamanho da Partícula , Fosforilação , Dobramento de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Sus scrofa , Difração de Raios XRESUMO
The molecular basis for the severity and rapid spread of the COVID-19 disease caused by SARS-CoV-2 is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04 Å resolution by x-ray crystallography. The structure reveals a ~60 residue core similar to SARS-CoV ORF7a with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate non-covalent interface is formed by another SARS-CoV-2-specific sequence, 73 YIDI 76 . Together the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.