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1.
Nature ; 591(7851): 677-681, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658720

RESUMO

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Glicina/química , Glicina/metabolismo , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Cristalografia , Humanos , Modelos Moleculares , Piperazinas/química , Piperazinas/farmacologia , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único , Sulfonas/química , Sulfonas/farmacologia , Síncrotrons
2.
J Biol Chem ; 299(5): 104627, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36944399

RESUMO

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.


Assuntos
Adesinas de Escherichia coli , Receptor de Manose , Modelos Moleculares , Humanos , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Manose/metabolismo , Receptor de Manose/química , Receptor de Manose/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Simulação de Acoplamento Molecular
3.
J Synchrotron Radiat ; 31(Pt 1): 186-194, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37971957

RESUMO

Here, high-throughput tomography (HiTT), a fast and versatile phase-contrast imaging platform for life-science samples on the EMBL beamline P14 at DESY in Hamburg, Germany, is presented. A high-photon-flux undulator beamline is used to perform tomographic phase-contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X-ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X-ray imaging data from potentially hundreds of samples during a beam-time shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. The successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin-embedded preparations is demonstrated. Furthermore, the feasibility of HiTT to be used as a targeting tool for volume electron microscopy, as well as using HiTT to study plant morphology, is demonstrated. It is also shown how the high-throughput nature of the work has allowed large numbers of `identical' samples to be imaged to enable statistically relevant sample volumes to be studied.


Assuntos
Robótica , Síncrotrons , Raios X , Tomografia Computadorizada por Raios X , Alemanha
4.
Genome Res ; 30(7): 962-973, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32703884

RESUMO

RNA-binding proteins (RBPs) regulate RNA metabolism at multiple levels by affecting splicing of nascent transcripts, RNA folding, base modification, transport, localization, translation, and stability. Despite their central role in RNA function, the RNA-binding specificities of most RBPs remain unknown or incompletely defined. To address this, we have assembled a genome-scale collection of RBPs and their RNA-binding domains (RBDs) and assessed their specificities using high-throughput RNA-SELEX (HTR-SELEX). Approximately 70% of RBPs for which we obtained a motif bound to short linear sequences, whereas ∼30% preferred structured motifs folding into stem-loops. We also found that many RBPs can bind to multiple distinctly different motifs. Analysis of the matches of the motifs in human genomic sequences suggested novel roles for many RBPs. We found that three cytoplasmic proteins-ZC3H12A, ZC3H12B, and ZC3H12C-bound to motifs resembling the splice donor sequence, suggesting that these proteins are involved in degradation of cytoplasmic viral and/or unspliced transcripts. Structural analysis revealed that the RNA motif was not bound by the conventional C3H1 RNA-binding domain of ZC3H12B. Instead, the RNA motif was bound by the ZC3H12B's PilT N terminus (PIN) RNase domain, revealing a potential mechanism by which unconventional RBDs containing active sites or molecule-binding pockets could interact with short, structured RNA molecules. Our collection containing 145 high-resolution binding specificity models for 86 RBPs is the largest systematic resource for the analysis of human RBPs and will greatly facilitate future analysis of the various biological roles of this important class of proteins.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Sequência de Bases , Genoma Humano , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Ribonucleases/química , Ribonucleases/metabolismo , Técnica de Seleção de Aptâmeros
5.
J Synchrotron Radiat ; 30(Pt 4): 723-738, 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37343017

RESUMO

The ability to utilize a hybrid-photon-counting detector to its full potential can significantly influence data quality, data collection speed, as well as development of elaborate data acquisition schemes. This paper facilitates the optimal use of EIGER2 detectors by providing theory and practical advice on (i) the relation between detector design, technical specifications and operating modes, (ii) the use of corrections and calibrations, and (iii) new acquisition features: a double-gating mode, 8-bit readout mode for increasing temporal resolution, and lines region-of-interest readout mode for frame rates up to 98 kHz. Examples of the implementation and application of EIGER2 at several synchrotron sources (ESRF, PETRA III/DESY, ELETTRA, AS/ANSTO) are presented: high accuracy of high-throughput data in serial crystallography using hard X-rays; suppressing higher harmonics of undulator radiation, improving peak shapes, increasing data collection speed in powder X-ray diffraction; faster ptychography scans; and cleaner and faster pump-and-probe experiments.


Assuntos
Fótons , Síncrotrons , Raios X , Radiografia , Difração de Raios X
6.
J Virol ; 96(1): e0150521, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34613791

RESUMO

During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, reduces protein size, influencing the origin and evolution of NLSs and NoLSs in viruses. IMPORTANCE Here, we investigated the molecular mechanism of nuclear localization signal (NLS) and nucleolar localization signal (NoLS) integration into the basic domain of HIV-1 Tat (49RKKRRQRRR57) and found that these two supplementary functions (i.e., function of NLS and function of NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.


Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/fisiologia , Sinais de Localização Nuclear , Domínios e Motivos de Interação entre Proteínas , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Sequência Consenso , Evolução Molecular , Interações Hospedeiro-Patógeno , Modelos Moleculares , Ligação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
7.
Cell ; 133(2): 328-39, 2008 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-18423203

RESUMO

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.


Assuntos
Glicoproteínas/metabolismo , Guanilato Quinases/química , Guanilato Quinases/metabolismo , Neuropeptídeos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cristalografia por Raios X , Humanos , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos
8.
Proc Natl Acad Sci U S A ; 117(37): 22841-22848, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32859757

RESUMO

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.

9.
Nat Methods ; 16(10): 979-982, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31527838

RESUMO

We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.


Assuntos
Cristalografia/métodos , Síncrotrons , Acetilglucosamina/química , Aldose-Cetose Isomerases/química , Glucose/química , Muramidase/química , Estudo de Prova de Conceito
10.
Proc Natl Acad Sci U S A ; 116(38): 18911-18916, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31462497

RESUMO

Marine polychaetes Odontosyllis undecimdonta, commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system's low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin.


Assuntos
Substâncias Luminescentes/química , Poliquetos/química , Animais , Vias Biossintéticas , Cor , Indóis/química , Indóis/metabolismo , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Proteínas Luminescentes/metabolismo , Estrutura Molecular , Oxirredução , Poliquetos/metabolismo , Pirazinas/química , Pirazinas/metabolismo
11.
J Synchrotron Radiat ; 26(Pt 4): 1208-1212, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31274445

RESUMO

A new ultra-compact transfocator (UCTF) based on X-ray compound refractive lenses (CRLs) is presented. The device can be used to change the number of one- and two-dimensional focusing CRLs by moving the individual parabolic lenses one-by-one independently, thus providing permanent energy and focal-length tunability for scanning and full-field X-ray microscopy applications. The small overall size and light weight of the device allow it to be integrated in any synchrotron beamline, while even simplifying the experimental layout. The UCTF was tested at the Excillium MetalJet microfocus X-ray source and at the P14 EMBL (PETRA-III) beamline, demonstrating high mechanical stability and lens positioning repeatability.

12.
J Synchrotron Radiat ; 26(Pt 3): 714-719, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31074435

RESUMO

Full-field X-ray imaging and microscopy with polymer compound refractive nano-lenses is demonstrated. Experiments were carried out at beamline ID13 at the European Synchrotron and yielded a resolution of 100 nm. The lenses were demonstrated to be functioning even after an absorbed dose of ∼107 Gy. This article also discusses issues related to lens aberrations, astigmatism and radiation stability, and thus ways of improving the lens further are considered. Polymer nano-lenses are versatile and are promissing for nano-focusing and compact X-ray microscopy.

13.
J Synchrotron Radiat ; 26(Pt 2): 393-405, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30855248

RESUMO

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.

14.
Proteins ; 86(9): 912-923, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29722065

RESUMO

RipA plays a vital role during cell division of Mycobacterium tuberculosis by degrading the cell wall peptidoglycan at the septum, allowing daughter cell separation. The peptidoglycan degrading activity relies on the NlpC/P60 domain, and as it is potentially harmful when deregulated, spatial and temporal control is necessary in this process. The N-terminal domain of RipA has been proposed to play an inhibitory role blocking the C-terminal NlpC/P60 domain. Accessibility of the active site cysteine residue is however not limited by the presence of the N-terminal domain, but by the lid-module of the inter-domain linker, which is situated in the peptide binding groove of the crystal structures of the catalytic domain. The 2.2 Å resolution structure of the N-terminal domain, determined by Se-SAD phasing, reveals an all-α-fold with 2 long α-helices, and shows similarity to bacterial periplasmic protein domains with scaffold-building role. Size exclusion chromatography and SAXS experiments are consistent with dimer formation of this domain in solution. The SAXS data from the periplasmic two-domain RipA construct suggest a rigid baton-like structure of the N-terminal module, with the catalytic domain connected by a 24 residue long flexible linker. This flexible linker allows for a catalytic zone, which is part of the spatiotemporal control of peptidoglycan degradation.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/enzimologia , Hidrolases/metabolismo , Proteínas de Bactérias/química , Biocatálise , Domínio Catalítico , Hidrolases/química , Mycobacterium tuberculosis/metabolismo , Peptidoglicano/metabolismo , Conformação Proteica , Multimerização Proteica
15.
J Synchrotron Radiat ; 24(Pt 1): 19-28, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28009543

RESUMO

High-quality high-multiplicity X-ray diffraction data were collected on five different crystals of thaumatin using a homogeneous-profile X-ray beam at E = 8 keV to investigate the counteracting effects of increased multiplicity and increased radiation damage on the quality of anomalous diffraction data collected on macromolecular crystals. By comparing sulfur substructures obtained from subsets of the data selected as a function of absorbed X-ray dose with sulfur positions in the respective refined reference structures, the doses at which the highest quality of anomalous differences could be obtained were identified for the five crystals. A statistic σ{ΔF}D, calculated as the width σ of the normalized distribution of a set {ΔF} of anomalous differences collected at a dose D, is suggested as a measure of anomalous data quality as a function of dose. An empirical rule is proposed to identify the dose at which the gains in data quality due to increased multiplicity are outbalanced by the losses due to decreases in signal-to-noise as a consequence of radiation damage. Identifying this point of diminishing returns allows the optimization of the choice of data collection parameters and the selection of data to be used in subsequent crystal structure determination steps.


Assuntos
Cristalografia por Raios X , Substâncias Macromoleculares , Coleta de Dados , Modelos Moleculares , Enxofre , Difração de Raios X
16.
J Synchrotron Radiat ; 24(Pt 1): 323-332, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28009574

RESUMO

The macromolecular crystallography P13 beamline is part of the European Molecular Biology Laboratory Integrated Facility for Structural Biology at PETRA III (DESY, Hamburg, Germany) and has been in user operation since mid-2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallographic data acquisition exploiting a wide range of elemental absorption edges for experimental phase determination. An adaptive Kirkpatrick-Baez focusing system provides an X-ray beam with a high photon flux and tunable focus size to adapt to diverse experimental situations. Data collections at energies as low as 4 keV (λ = 3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux particularly at low energy (up to 1011 photons s-1 at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4 × 1011 photons s-1 at 17.5 keV) combined with a large area detector mounted on a 2θ arm allows data collection to sub-atomic resolution (0.55 Å). A peak flux of about 8.0 × 1012 photons s-1 is reached at 11 keV. Automated sample mounting is available by means of the robotic sample changer `MARVIN' with a dewar capacity of 160 samples. In close proximity to the beamline, laboratories have been set up for sample preparation and characterization; a laboratory specifically equipped for on-site heavy atom derivatization with a library of more than 150 compounds is available to beamline users.

17.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 11): 2328-43, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26527148

RESUMO

Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.


Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Animais , Bacillus/química , Bacteriorodopsinas/química , Bombyx/química , Análise por Conglomerados , Cristalização/métodos , Halobacterium salinarum/química , Proteínas de Insetos/química , Modelos Moleculares , Muramidase/química , Proteínas de Plantas/química , Plantas/química , Síncrotrons , Termolisina/química , Fluxo de Trabalho
18.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 11): 2344-53, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26527149

RESUMO

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.


Assuntos
Proteínas de Bactérias/química , Oxigenases/química , Pseudomonas putida/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxigenases/genética , Oxigenases/metabolismo , Plasmídeos/genética , Conformação Proteica , Dobramento de Proteína , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Alinhamento de Sequência
19.
Chembiochem ; 16(1): 110-8, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25393087

RESUMO

Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.


Assuntos
Acetolactato Sintase/química , Bacillus subtilis/química , Proteínas de Bactérias/química , Butanóis/química , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Bacillus subtilis/classificação , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Biocombustíveis , Butanóis/metabolismo , Domínio Catalítico , Cátions Bivalentes , Coenzimas/química , Coenzimas/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hemiterpenos , Cetoácidos/química , Cetoácidos/metabolismo , Lactatos/química , Lactatos/metabolismo , Magnésio/química , Magnésio/metabolismo , Modelos Moleculares , Filogenia , Mutação Puntual , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Relação Estrutura-Atividade , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismo
20.
Nat Commun ; 15(1): 1709, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38402242

RESUMO

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by in vitro crystallization extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely native proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, improved strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.


Assuntos
Lasers , Síncrotrons , Cristalografia por Raios X , Cristalização , Proteínas Recombinantes/genética
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