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1.
Acta Crystallogr D Struct Biol ; 80(Pt 5): 350-361, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38682668

RESUMO

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.


Assuntos
Listeria monocytogenes , Listeria monocytogenes/enzimologia , Cristalografia por Raios X/métodos , Sítios de Ligação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Fosfatos de Dinucleosídeos/metabolismo , Fosfatos de Dinucleosídeos/química , Antibacterianos/farmacologia , Humanos , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Conformação Proteica
2.
ChemMedChem ; 19(9): e202400057, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38385828

RESUMO

A 1H-isoindol-3-amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin-like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine-derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate-analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine-derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin-dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas-related G-protein coupled receptor GPCR-X2 could be a potential off-target for certain benzamidine-derived furin inhibitors.


Assuntos
Antivirais , Desenho de Fármacos , Furina , Furina/antagonistas & inibidores , Furina/metabolismo , Humanos , Antivirais/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Estrutura-Atividade , Células A549 , Vírus da Influenza A/efeitos dos fármacos , Cristalografia por Raios X , Indóis/farmacologia , Indóis/química , Indóis/síntese química , Estrutura Molecular , Modelos Moleculares , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Relação Dose-Resposta a Droga
3.
ACS Chem Biol ; 19(2): 563-574, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38232960

RESUMO

The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.


Assuntos
Descoberta de Drogas , SARS-CoV-2 , Descoberta de Drogas/métodos , SARS-CoV-2/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância Magnética , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/metabolismo , Antivirais/farmacologia , Simulação de Acoplamento Molecular
4.
ACS Omega ; 8(48): 46051-46065, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38075755

RESUMO

The Shigella pathogenicity factor IpgC belongs to the class II of type III secretion system chaperones, whose members are characterized by a tetratricopeptide repeat (TPR) domain consisting of three and a half TPR motifs. Since IpgC is essential for Shigella virulence, we determined a high-resolution crystal structure of this chaperone to facilitate its use as a target for the structure-based design of anti-shigellosis compounds. The crystal structure revealed two possible homodimer assemblies, which strongly differ from the homodimer architectures so far known for IpgC and orthologues thereof. Through crystallographic fragment screening, we identified 10 small molecules that bind to IpgC and, therefore, are available for expansion to generate larger, more potent binders. A follow-up compound, based on one of our fragment hits, binds to a strictly conserved site, which overlaps with the binding site of the chaperone's substrates, IpaB and IpaC. Therefore, it constitutes a promising starting point for the design of functional IpgC inhibitors.

5.
Acta Crystallogr D Struct Biol ; 79(Pt 9): 857-865, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37574972

RESUMO

The increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/metabolismo , Mycobacterium tuberculosis/metabolismo , Sítios de Ligação , Desenho de Fármacos
6.
Chemistry ; 29(23): e202203967, 2023 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-36799129

RESUMO

The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.


Assuntos
Neoplasias Colorretais , Pirazóis , Humanos , Pirazóis/química , Pirimidinas/farmacologia , Pirimidinas/química , Linhagem Celular , Neoplasias Colorretais/tratamento farmacológico , Linhagem Celular Tumoral
7.
Protein Sci ; 32(1): e4542, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36519786

RESUMO

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.


Assuntos
DNA (Citosina-5-)-Metiltransferases , DNA Metiltransferase 3A , Proteína 2 de Ligação a Metil-CpG , Sítios de Ligação , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Histonas/química , Histonas/metabolismo , Humanos
8.
J Med Chem ; 65(21): 14630-14641, 2022 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-36260741

RESUMO

The identification of starting points for compound development is one of the key steps in early-stage drug discovery. Information-rich techniques such as crystallographic fragment screening can potentially increase the efficiency of this step by providing the structural information of the binding mode of the ligands in addition to the mere binding information. Here, we present the crystallographic screening of our 1000-plus-compound F2X-Universal Library against the complex of the yeast spliceosomal Prp8 RNaseH-like domain and the snRNP assembly factor Aar2. The observed 269 hits are distributed over 10 distinct binding sites on the surface of the protein-protein complex. Our work shows that hit clusters from large-scale crystallographic fragment screening campaigns identify known interaction sites with other proteins and suggest putative additional interaction sites. Furthermore, the inherent binding pose validation within the hit clusters may accelerate downstream compound optimization.


Assuntos
Descoberta de Drogas , Proteínas , Cristalografia por Raios X , Ligantes , Descoberta de Drogas/métodos , Sítios de Ligação , Ligação Proteica
9.
Protein Sci ; 31(9): e4391, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36040268

RESUMO

In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.


Assuntos
Proteínas , Cristalografia por Raios X , Ligantes , Proteínas/química
10.
J Mol Biol ; 434(16): 167720, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35839840

RESUMO

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.


Assuntos
ADP-Ribosilação , Adenosina/análogos & derivados , Inibidores de Protease de Coronavírus , Poli(ADP-Ribose) Polimerases , SARS-CoV-2 , ADP-Ribosilação/efeitos dos fármacos , Adenosina/química , Adenosina/farmacologia , Adenosina Difosfato Ribose/química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Humanos , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
11.
Acta Crystallogr D Struct Biol ; 77(Pt 9): 1168-1182, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34473087

RESUMO

In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure-based hit-to-lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow-up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof-of-concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow-up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single-digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X-ray crystallography.


Assuntos
Ácido Aspártico Endopeptidases , Cristalografia por Raios X/métodos , Descoberta de Drogas/métodos , Ligantes , Modelos Moleculares , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Domínio Catalítico , Ligação Proteica
12.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34290142

RESUMO

Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5' RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggesting that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies.


Assuntos
Difosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , RNA Helicases DEAD-box/metabolismo , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Sítios de Ligação , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformação Proteica
13.
Acta Crystallogr D Struct Biol ; 77(Pt 6): 799-808, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34076593

RESUMO

Crystallographic fragment screening (CFS) has become one of the major techniques for screening compounds in the early stages of drug-discovery projects. Following the advances in automation and throughput at modern macromolecular crystallography beamlines, the bottleneck for CFS has shifted from collecting data to organizing and handling the analysis of such projects. The complexity that emerges from the use of multiple methods for processing and refinement and to search for ligands requires an equally sophisticated solution to summarize the output, allowing researchers to focus on the scientific questions instead of on software technicalities. FragMAXapp is the fragment-screening project-management tool designed to handle CFS projects at MAX IV Laboratory. It benefits from the powerful computing infrastructure of large-scale facilities and, as a web application, it is accessible from everywhere.


Assuntos
Descoberta de Drogas/métodos , Ligantes , Substâncias Macromoleculares/química , Modelos Moleculares , Proteínas/química , Software , Análise de Dados
14.
J Appl Crystallogr ; 54(Pt 1): 376-382, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33833659

RESUMO

In the past two decades, most of the steps in a macromolecular crystallography experiment have undergone tremendous development with respect to speed, feasibility and increase of throughput. The part of the experimental workflow that is still a bottleneck, despite significant efforts, involves the manipulation and harvesting of the crystals for the diffraction experiment. Here, a novel low-cost device is presented that functions as a cover for 96-well crystallization plates. This device enables access to the individual experiments one at a time by its movable parts, while minimizing evaporation of all other experiments of the plate. In initial tests, drops of many typically used crystallization cocktails could be successfully protected for up to 6 h. Therefore, the manipulation and harvesting of crystals is straightforward for the experimenter, enabling significantly higher throughput. This is useful for many macromolecular crystallography experiments, especially multi-crystal screening campaigns.

15.
J Vis Exp ; (169)2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33749678

RESUMO

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results. Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY  II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input. Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.


Assuntos
Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Berlim , Cristalização , Coleta de Dados , Ligantes , Proteínas/química , Software , Síncrotrons , Fluxo de Trabalho
16.
Acta Crystallogr D Struct Biol ; 76(Pt 11): 1065-1079, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33135678

RESUMO

This study focuses on the polymorphism of human insulin (HI) upon the binding of the phenolic derivatives p-coumaric acid or trans-resveratrol over a wide pH range. The determination of the structural behaviour of HI via X-ray powder diffraction (XRPD) and single-crystal X-ray diffraction (SCXRD) is reported. Four distinct polymorphs were identified, two of which have not been reported previously. The intermediate phase transitions are discussed. One of the novel monoclinic polymorphs displays the highest molecular packing among insulin polymorphs of the same space group to date; its structure was elucidated by SCXRD. XRPD data collection was performed using a variety of instrumental setups and a systematic comparison of the acquired data is presented. A laboratory diffractometer was used for screening prior to high-resolution XRPD data collection on the ID22 beamline at the European Synchrotron Radiation Facility. Additional measurements for the most representative samples were performed on the X04SA beamline at the Swiss Light Source (SLS) using the MYTHEN II detector, which allowed the detection of minor previously untraceable impurities and dramatically improved the d-spacing resolution even for poorly diffracting samples.


Assuntos
Ácidos Cumáricos , Insulina Regular Humana , Modelos Moleculares , Resveratrol , Ácidos Cumáricos/química , Cristalização , Humanos , Insulina Regular Humana/química , Substâncias Macromoleculares , Difração de Pó , Ligação Proteica , Resveratrol/química , Difração de Raios X
17.
Structure ; 28(6): 694-706.e5, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32413289

RESUMO

Crystallographic fragment screening (CFS) provides excellent starting points for projects concerned with drug discovery or biochemical tool compound development. One of the fundamental prerequisites for effective CFS is the availability of a versatile fragment library. Here, we report on the assembly of the 1,103-compound F2X-Universal Library and its 96-compound sub-selection, the F2X-Entry Screen. Both represent the available fragment chemistry and are highly diverse in terms of their 3D-pharmacophore variations. Validation of the F2X-Entry Screen in CFS campaigns using endothiapepsin and the Aar2/RNaseH complex yielded hit rates of 30% and 21%, respectively, and revealed versatile binding sites. Dry presentation of the libraries allows CFS campaigns to be carried out with or without the co-solvent DMSO present. Most of the hits in our validation campaigns could be reproduced also in the absence of DMSO. Consequently, CFS can be carried out more efficiently and for a wider range of conditions and targets.


Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Compostos Químicos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química
18.
Biophys J ; 114(4): 788-799, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29490241

RESUMO

Precursor messenger RNA splicing is mediated by the spliceosome, a large and dynamic molecular machine composed of five small nuclear RNAs and numerous proteins. Many spliceosomal proteins are predicted to be intrinsically disordered or contain large disordered regions, but experimental validation of these predictions is scarce, and the precise functions of these proteins are often unclear. Here, we show via circular dichroism spectroscopy, dynamic light scattering, and NMR spectroscopy that the yeast spliceosomal disassembly factor Ntr2 is largely intrinsically disordered. Peptide SPOT analyses, analytical size-exclusion chromatography, and surface plasmon resonance measurements revealed that Ntr2 uses an N-terminal region to bind the C-terminal helicase unit of the Brr2 RNA helicase, an enzyme involved in spliceosome activation and implicated in splicing catalysis and spliceosome disassembly. NMR analyses suggested that Ntr2 does not adopt a tertiary structure and likely remains disordered upon complex formation. RNA binding and unwinding studies showed that Ntr2 downregulates Brr2 helicase activity in vitro by modulating the fraction of helicase molecules productively bound to the RNA substrate. Our data clarify the nature of a physical link between Brr2 and Ntr2, and point to the possibility of a functional Ntr2-Brr2 interplay during splicing.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , RNA Helicases/metabolismo , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Catálise , Proteínas Intrinsicamente Desordenadas/química , RNA Helicases/química , Proteínas de Saccharomyces cerevisiae/química
19.
Antioxid Redox Signal ; 29(7): 615-636, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29237286

RESUMO

AIMS: Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections, which requires efficient protection mechanisms to avoid destruction. Here, we have investigated the changes in the RNA-seq transcriptome by the strong oxidant sodium hypochlorite (NaOCl) in S. aureus USA300 to identify novel redox-sensing mechanisms that provide protection under infection conditions. RESULTS: NaOCl stress caused an oxidative stress response in S. aureus as indicated by the induction of the PerR, QsrR, HrcA, and SigmaB regulons in the RNA-seq transcriptome. The hypR-merA (USA300HOU_0588-87) operon was most strongly upregulated under NaOCl stress, which encodes for the Rrf2-family regulator HypR and the pyridine nucleotide disulfide reductase MerA. We have characterized HypR as a novel redox-sensitive repressor that controls MerA expression and directly senses and responds to NaOCl and diamide stress via a thiol-based mechanism in S. aureus. Mutational analysis identified Cys33 and the conserved Cys99 as essential for NaOCl sensing, while Cys99 is also important for repressor activity of HypR in vivo. The redox-sensing mechanism of HypR involves Cys33-Cys99 intersubunit disulfide formation by NaOCl stress both in vitro and in vivo. Moreover, the HypR-controlled flavin disulfide reductase MerA was shown to protect S. aureus against NaOCl stress and increased survival in J774A.1 macrophage infection assays. Conclusion and Innovation: Here, we identified a new member of the widespread Rrf2 family as redox sensor of NaOCl stress in S. aureus that uses a thiol/disulfide switch to regulate defense mechanisms against the oxidative burst under infections in S. aureus. Antioxid. Redox Signal. 29, 615-636.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Ácido Hipocloroso/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Biologia Computacional , Camundongos , Modelos Moleculares , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo
20.
Cell Cycle ; 16(1): 100-112, 2017 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-27880071

RESUMO

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.


Assuntos
RNA Helicases/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Spliceossomos/metabolismo , Sequência de Aminoácidos , Chaetomium , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Helicases/química , RNA Fúngico/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Homologia Estrutural de Proteína
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