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1.
Nat Commun ; 11(1): 4916, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004803

RESUMO

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.


Assuntos
Brassica rapa/fisiologia , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Animais , Cristalografia , Flores/metabolismo , Haplótipos , Simulação de Dinâmica Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Pólen/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera
2.
Structure ; 28(10): 1084-1086, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33027690

RESUMO

The determination of protein structures from nanocrystals grown in lipidic cubic phase (LCP) is a promising crystallographic approach. In this issue of Structure, Zhu et al. (2020) extract crystals from the dense matrix of monoolein LCP for interrogation by micro electron diffraction (MicroED) and yield a 2 Å structure of Proteinase K.


Assuntos
Elétrons , Lipídeos , Cristalografia , Proteínas
3.
Nat Commun ; 11(1): 4720, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948753

RESUMO

Cellulose, the most abundant biopolymer on earth, is a versatile, energy rich material found in the cell walls of plants, bacteria, algae, and tunicates. It is well established that cellulose is crystalline, although the orientational order of cellulose crystallites normal to the plane of the cell wall has not been characterized. A preferred orientational alignment of cellulose crystals could be an important determinant of the mechanical properties of the cell wall and of cellulose-cellulose and cellulose-matrix interactions. Here, the crystalline structures of cellulose in primary cell walls of onion (Allium cepa), the model eudicot Arabidopsis (Arabidopsis thaliana), and moss (Physcomitrella patens) were examined through grazing incidence wide angle X-ray scattering (GIWAXS). We find that GIWAXS can decouple diffraction from cellulose and epicuticular wax crystals in cell walls. Pole figures constructed from a combination of GIWAXS and X-ray rocking scans reveal that cellulose crystals have a preferred crystallographic orientation with the (200) and (110)/([Formula: see text]) planes preferentially stacked parallel to the cell wall. This orientational ordering of cellulose crystals, termed texturing in materials science, represents a previously unreported measure of cellulose organization and contradicts the predominant hypothesis of twisting of microfibrils in plant primary cell walls.


Assuntos
Parede Celular/química , Celulose/química , Plantas/química , Arabidopsis/química , Bryopsida/química , Cristalografia , Cristalografia por Raios X , Microfibrilas/química
4.
Nat Commun ; 11(1): 4511, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908128

RESUMO

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.


Assuntos
Cristalografia/instrumentação , Elétrons , Dispositivos Lab-On-A-Chip , Lasers , Aldeído Liases/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Hidrodinâmica
5.
Nat Commun ; 11(1): 4851, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978386

RESUMO

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Ligantes , Engenharia Metabólica/métodos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Cristalografia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Lisina/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Termodinâmica
6.
Int J Mol Sci ; 21(14)2020 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-32664570

RESUMO

A dodecadepsipeptide valinomycin (VLM) has been most recently reported to be a potential anti-coronavirus drug that could be efficiently produced on a large scale. It is thus of importance to study solid-phase forms of VLM in order to be able to ensure its polymorphic purity in drug formulations. The previously available solid-state NMR (SSNMR) data are combined with the plane-wave DFT computations in the NMR crystallography framework. Structural/spectroscopical predictions (the PBE functional/GIPAW method) are obtained to characterize four polymorphs of VLM. Interactions which confer a conformational stability to VLM molecules in these crystalline forms are described in detail. The way how various structural factors affect the values of SSNMR parameters is thoroughly analyzed, and several SSNMR markers of the respective VLM polymorphs are identified. The markers are connected to hydrogen bonding effects upon the corresponding (13C/15N/1H) isotropic chemical shifts of (CO, Namid, Hamid, Hα) VLM backbone nuclei. These results are expected to be crucial for polymorph control of VLM and in probing its interactions in dosage forms.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Valinomicina/química , Betacoronavirus/química , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Isótopos de Carbono/química , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Cristalografia , Ligação de Hidrogênio , Isótopos de Nitrogênio/química , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Valinomicina/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(31): 18224-18230, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32680959

RESUMO

Utilizing an ionic liquid strategy, we report crystal structures of salts of free anionic nucleobases and base pairs previously studied only computationally and in the gas phase. Reaction of tetrabutylammonium ([N4444]+) or tetrabutylphosphonium ([P4444]+) hydroxide with adenine (HAd) and thymine (HThy) led to hydrated salts of deprotonated adenine, [N4444][Ad]·2H2O, and thymine, [P4444][Thy]·2H2O, as well as the double salt cocrystal, [P4444]2[Ad][Thy]·3H2O·2HThy. The cocrystal includes the anionic [Ad-(HThy)] base pair which is a stable formation in the solid state that has previously not even been suggested. It exhibits Watson-Crick connectivity as found in DNA but which is unusual for the free neutral base pairs. The stability of the observed anionic bases and their supramolecular formations and hydrates has also been examined by electronic structure calculations, contributing to more insight into how base pairs can bind when a proton is removed and highlighting mechanisms of stabilization or chemical transformation in the DNA chains.


Assuntos
Adenina/química , Pareamento de Bases , Timina/química , Ânions , Cristalografia , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico
8.
Proc Natl Acad Sci U S A ; 117(30): 18059-18067, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669442

RESUMO

Orexins are neuropeptides that activate the rhodopsin-like G protein-coupled receptors OX1R and OX2R. The orexin system plays an important role in the regulation of the sleep-wake cycle and the regulation of feeding and emotions. The nonselective orexin receptor antagonist suvorexant has been the first drug on the market targeting the orexin system and is prescribed for the treatment of insomnia. Subtype-selective OX1R antagonists are valuable tools to further investigate the functions and physiological role of the OX1R in vivo and promising lead compounds for the treatment of drug addiction, anxiety, pain or obesity. Starting from the OX1R and OX2R crystal structures bound to suvorexant, we exploited a single amino acid difference in the orthosteric binding site by using molecular docking and structure-based drug design to optimize ligand interactions with the OX1R while introducing repulsive interactions with the OX2R. A newly established enantiospecific synthesis provided ligands showing up to 75-fold selectivity for the OX1R over the OX2R subtype. The structure of a new OX1R antagonist with subnanomolar affinity (JH112) was determined by crystallography in complex with the OX1R and corresponded closely to the docking-predicted geometry. JH112 exhibits high selectivity over a panel of different GPCRs, is able to cross the blood-brain barrier and acts as slowly diffusing and insurmountable antagonist for Gq protein activation and in particular ß-arrestin-2 recruitment at OX1R. This study demonstrates the potential of structure-based drug design to develop more subtype-selective GPCR ligands with potentially reduced side effects and provides an attractive probe molecule and lead compound.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Sítios de Ligação , Cristalografia , Desenho de Fármacos , Cinética , Ligantes , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Relação Estrutura-Atividade
9.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1021-1030, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597053

RESUMO

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel ß-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.


Assuntos
Hidrolases de Éster Carboxílico , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Cristalografia , Pectinas/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato
10.
Nature ; 583(7815): 314-318, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499654

RESUMO

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.


Assuntos
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sítios de Ligação , Cristalografia , Elétrons , Transporte de Íons , Isomerismo , Lasers , Prótons , Teoria Quântica , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Sódio/metabolismo , Análise Espectral , Eletricidade Estática , Fatores de Tempo
11.
PLoS One ; 15(5): e0232540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374767

RESUMO

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.


Assuntos
Apoferritinas/química , Apoferritinas/ultraestrutura , Microscopia Crioeletrônica/métodos , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica/instrumentação , Cristalografia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Subunidades Proteicas , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Eletricidade Estática
12.
Nat Commun ; 11(1): 1663, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245964

RESUMO

Massively parallel, quantitative measurements of biomolecular activity across sequence space can greatly expand our understanding of RNA sequence-function relationships. We report the development of an RNA-array assay to perform such measurements and its application to a model RNA: the core glmS ribozyme riboswitch, which performs a ligand-dependent self-cleavage reaction. We measure the cleavage rates for all possible single and double mutants of this ribozyme across a series of ligand concentrations, determining kcat and KM values for active variants. These systematic measurements suggest that evolutionary conservation in the consensus sequence is driven by maintenance of the cleavage rate. Analysis of double-mutant rates and associated mutational interactions produces a structural and functional mapping of the ribozyme sequence, revealing the catalytic consequences of specific tertiary interactions, and allowing us to infer structural rearrangements that permit certain sequence variants to maintain activity.


Assuntos
Proteínas de Bactérias/genética , Evolução Molecular , RNA Catalítico/genética , Riboswitch/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência Consenso/genética , Cristalografia , Ensaios Enzimáticos , Sequenciamento de Nucleotídeos em Larga Escala , Ligantes , Mutação , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/metabolismo , Análise de Sequência de RNA , Relação Estrutura-Atividade
13.
J Phys Chem A ; 124(16): 3109-3119, 2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32233483

RESUMO

The principal components of the 13C chemical shift tensors for the ten crystallographically distinct carbon atoms of the active pharmaceutical ingredient cimetidine Form A have been measured using the FIREMAT technique. Density functional theory (DFT) calculations of 13C and 15N magnetic shielding tensors are used to assign the 13C and 15N peaks. DFT calculations were performed on cimetidine and a training set of organic crystals using both plane-wave and cluster-based approaches. The former set of calculations allowed several structural refinement strategies to be employed, including calculations utilizing a dispersion-corrected force field that was parametrized using 13C and 15N magnetic shielding tensors. The latter set of calculations featured the use of resource-intensive hybrid-DFT methods for the calculation of magnetic shielding tensors. Calculations on structures refined using the new force-field correction result in improved values of 15N magnetic shielding tensors (as gauged by agreement with experimental chemical shift tensors), although little improvement is seen in the prediction of 13C shielding tensors. Calculations of 13C and 15N magnetic shielding tensors using hybrid functionals show better agreement with experimental values in comparison to those using GGA functionals, independent of the method of structural refinement; the shielding of carbon atoms bonded to nitrogen are especially improved using hybrid DFT methods.


Assuntos
Cimetidina/química , Teoria da Densidade Funcional , Isótopos de Carbono , Cristalografia , Espectroscopia de Ressonância Magnética/normas , Estrutura Molecular , Padrões de Referência
14.
Molecules ; 25(4)2020 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-32102204

RESUMO

The four compounds, namely: 5-nitro-2-furaldehyde thiosemicarbazone (1); 5-nitro-2-thiophene thiosemicarbazone (2); 5-nitro-2-furaldehyde semicarbazone (3); and 5-nitro-2-thiophene semicarbazone (4) were synthesized and crystallized. The three new crystal structures of 1, 2, and 4 were determined and compared to three already known crystal structures of 3. Additionally, two new polymorphic forms of 1 solvate were synthesized and studied. The influence of the exchange of 2-thiophene to 2-furaldehyde as well as thiosemicarbazone and semicarbazone on the self-assembly of supramolecular nets was elucidated and discussed in terms of the formed synthons and assemblies accompanied by Full Interaction Maps analysis. Changes in the strength of IR oscillators caused by the molecular and crystal packing effects are described and explained in terms of changes of electron density.


Assuntos
Tiossemicarbazonas/química , Cristalografia , Cristalografia por Raios X , Conformação Molecular , Estrutura Molecular , Espectrofotometria Infravermelho , Tiofenos/química , Tiossemicarbazonas/síntese química
15.
Nat Commun ; 11(1): 996, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081905

RESUMO

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.


Assuntos
Cristalografia/métodos , Proteínas/química , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Muramidase/química , Muramidase/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Proteínas de Matriz de Corpos de Inclusão/química , Proteínas de Matriz de Corpos de Inclusão/ultraestrutura , Tamanho da Partícula , Conformação Proteica , Proteínas/ultraestrutura
16.
AAPS PharmSciTech ; 21(3): 96, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103355

RESUMO

The true density of an amorphous solid is an important parameter for studying and modeling materials behavior. Experimental measurements of density using helium pycnometry are standard but may be prevented if the material is prone to rapid recrystallization, or preparation of gram quantities of reproducible pure component amorphous materials proves impossible. The density of an amorphous solid can be approximated by assuming it to be 95% of its respective crystallographic density; however, this can be inaccurate or impossible if the crystal structure is unknown. Molecular dynamic simulations were used to predict the density of 20 amorphous solid materials. The calculated density values for 10 amorphous solids were compared with densities that were experimentally determined using helium pycnometry. In these cases, the amorphous densities calculated using molecular dynamics had an average percent error of - 0.7% relative to the measured values, with a maximum error of - 3.48%. In contrast, comparisons of amorphous density approximated from crystallographic structures with pycnometrically measured values resulted in an average percent error of + 3.7%, with a maximum error of + 9.42%. These data suggest that the density of an amorphous solid can be accurately predicted using molecular dynamic simulations and allowed reliable calculation of density for the remaining 10 materials for which pycnometry could not be done.


Assuntos
Cristalografia/métodos , Compostos Heterocíclicos/química , Simulação de Dinâmica Molecular , Cristalografia/tendências , Previsões , Compostos Heterocíclicos/análise , Simulação de Dinâmica Molecular/tendências
17.
Proc Natl Acad Sci U S A ; 117(8): 4071-4077, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041886

RESUMO

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.


Assuntos
Cobre/química , Cristalografia/métodos , Geobacillus/enzimologia , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Domínio Catalítico , Cristalização , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Geobacillus/genética , Geobacillus/metabolismo , Modelos Moleculares , Nitrito Redutases/genética , Conformação Proteica
18.
Biochem J ; 477(3): 615-628, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31922182

RESUMO

Agrobacterium tumefaciens pathogens use specific compounds denoted opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ATP-binding cassette transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Structural and affinity data on mannopinic acid bound to SBPs are currently lacking while those of the three others mannityl opines are available. We investigated the molecular basis of two pathways for mannopinic acid uptake. MoaA was proposed as the specific SBP for mannopinic acid import in mannityl opines-assimilating agrobacteria, which was validated here using genetic studies and affinity measurements. We structurally characterized the mannopinic acid-binding mode of MoaA in two crystal forms at 2.05 and 1.57 Šresolution. We demonstrated that the non-specific SBP MotA, so far characterized as mannopine and Amadori compound importer, was also able to transport mannopinic acid. The structure of MotA bound to mannopinic acid at 2.2 Šresolution defines a different mannopinic acid-binding signature, similar to that of mannopine. Combining in vitro and in vivo approaches, this work allowed us to complete the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development.


Assuntos
Transporte Biológico , Proteínas de Transporte , Manitol/análogos & derivados , Tumores de Planta/microbiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia , Genes Bacterianos , Interações entre Hospedeiro e Microrganismos , Manitol/química , Manitol/metabolismo , Oxazinas/metabolismo
19.
Acta Crystallogr D Struct Biol ; 76(Pt 1): 1-8, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31909738

RESUMO

The conventional approach to search-model identification in molecular replacement (MR) is to screen a database of known structures using the target sequence. However, this strategy is not always effective, for example when the relationship between sequence and structural similarity fails or when the crystal contents are not those expected. An alternative approach is to identify suitable search models directly from the experimental data. SIMBAD is a sequence-independent MR pipeline that uses either a crystal lattice search or MR functions to directly locate suitable search models from databases. The previous version of SIMBAD used the fast AMoRe rotation-function search. Here, a new version of SIMBAD which makes use of Phaser and its likelihood scoring to improve the sensitivity of the pipeline is presented. It is shown that the additional compute time potentially required by the more sophisticated scoring is counterbalanced by the greater sensitivity, allowing more cases to trigger early-termination criteria, rather than running to completion. Using Phaser solved 17 out of 25 test cases in comparison to the ten solved with AMoRe, and it is shown that use of ensemble search models produces additional performance benefits.


Assuntos
Modelos Moleculares , Proteínas/química , Software , Cristalografia/métodos , Bases de Dados de Proteínas , Conformação Proteica
20.
Biochem Biophys Res Commun ; 524(1): 217-223, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31983429

RESUMO

Ferritins are ubiquitous iron-binding proteins that are mainly related to iron storage, detoxification and innate immunity. Here, we present the crystal structure of a marine invertebrate ferritin from Sinonovacula constricta at a resolution of 1.98 Å. The S. constricta ferritin (ScFer) possessed some structural similarities with vertebrate ferritins, and they shared a well-conserved architecture composed of five α-helical bundles that assembled into a cage-like structure with 24-subunits. The structure of ScFer also showed iron binding sites in the 3-fold channel, ferroxidase center, and putative nucleation sites. Further, electrostatic potential calculations suggested that the electrostatic gradient of the 3-fold channel could provide a guidance mechanism for iron entering the ferritin cavity.


Assuntos
Bivalves/metabolismo , Ferritinas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia , Ferritinas/ultraestrutura , Ferro/metabolismo , Filogenia , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade Estática
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