Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 14(1): 7718, 2023 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-38001074

RESUMO

Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction. In addition, we demonstrate that FimI contributes to anchoring the pilus to the outer membrane and report the crystal structures of (i) FimI in complex with the assembly chaperone FimC, (ii) the FimI-FimC complex bound to the N-terminal domain of FimD, and (iii) a ternary complex between FimI, FimA and FimC that provides structural insights on pilus assembly termination and pilus anchoring by FimI.


Assuntos
Proteínas de Escherichia coli , Fímbrias Bacterianas , Fímbrias Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/química , Escherichia coli/genética , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo
2.
Nat Commun ; 12(1): 5293, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489448

RESUMO

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , DNA Bacteriano/química , DnaB Helicases/química , Helicobacter pylori/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/química , Compostos de Alumínio/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Fluoretos/química , Fluoretos/metabolismo , Expressão Gênica , Helicobacter pylori/genética , Hidrólise , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
3.
J Biomol NMR ; 75(6-7): 255-272, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34170475

RESUMO

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.


Assuntos
Capsídeo/química , Isótopos de Carbono , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Prótons
4.
Angew Chem Int Ed Engl ; 60(10): 5339-5347, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33205864

RESUMO

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.


Assuntos
Hepacivirus/química , Bicamadas Lipídicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Bicamadas Lipídicas/química , Ressonância Magnética Nuclear Biomolecular , Fosfatidiletanolaminas/química , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica , Espectroscopia de Prótons por Ressonância Magnética , Proteínas não Estruturais Virais/química
5.
Proc Natl Acad Sci U S A ; 117(13): 7159-7170, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32179686

RESUMO

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA-protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch-mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.


Assuntos
RNA Helicases/metabolismo , Proteínas Repressoras/metabolismo , Adenosina Trifosfatases/metabolismo , Células HEK293 , Humanos , Conformação Proteica , Domínios Proteicos , RNA/metabolismo , RNA Helicases/química , Spliceossomos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...