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1.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 12): 725-730, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31797813

RESUMO

The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 Šresolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = ß = γ = 90°.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus aureus/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Conformação Proteica , Homologia de Sequência , Sistemas de Secreção Tipo VII/química , Sistemas de Secreção Tipo VII/genética , Sistemas de Secreção Tipo VII/isolamento & purificação
2.
Nature ; 576(7786): 321-325, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597161

RESUMO

Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems1. A molecular understanding of the type VII secretion mechanism has been hampered owing to a lack of three-dimensional structures of the fully assembled secretion apparatus. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/type VII secretion system from Mycobacterium smegmatis. The core of the ESX-3 secretion machine consists of four protein components-EccB3, EccC3, EccD3 and EccE3, in a 1:1:2:1 stoichiometry-which form two identical protomers. The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The domain of unknown function (DUF) adjacent to the stalk is identified as an ATPase domain that is essential for secretion. EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain. This suggests that conformational changes in the stalk domain-triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF-could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of type VII secretion systems differs markedly from that of other known secretion machines2, and provide a structural understanding of these systems that will be useful for the design of antimicrobial strategies that target bacterial virulence.


Assuntos
Microscopia Crioeletrônica , Mycobacterium smegmatis/química , Sistemas de Secreção Tipo VII/química , Sistemas de Secreção Tipo VII/ultraestrutura , Actinobacteria/química , Actinobacteria/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/ultraestrutura , Domínios Proteicos , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Relação Estrutura-Atividade , Thermomonospora , Sistemas de Secreção Tipo VII/isolamento & purificação
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