RESUMO
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.
Assuntos
Proteínas Arqueais/química , Proteínas Quimiotáticas Aceptoras de Metil/química , Pyrococcus horikoshii/química , Archaea/química , Archaea/genética , Archaea/metabolismo , Quimiotaxia/genética , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Flagelos/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/biossíntese , Proteínas Quimiotáticas Aceptoras de Metil/genética , Conformação Proteica , Multimerização Proteica , Pyrococcus horikoshii/genética , Pyrococcus horikoshii/metabolismo , Transdução de SinaisRESUMO
While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungal FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.
Assuntos
Corynebacterium/enzimologia , Ácido Graxo Sintase Tipo I/química , Cristalização , Ácido Graxo Sintase Tipo I/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Difração de Raios XRESUMO
Antibiotic therapy in response to Mycobacterium tuberculosis infections targets de novo fatty acid biosynthesis, which is orchestrated by a 1.9 MDa type I fatty acid synthase (FAS). Here, we characterize M. tuberculosis FAS by single-particle cryo-electron microscopy and interpret the data by docking the molecular models of yeast and Mycobacterium smegmatis FAS. Our analysis reveals a porous barrel-like structure of considerable conformational variability that is illustrated by the identification of several conformational states with altered topology in the multienzymatic assembly. This demonstrates that the barrel-like structure of M. tuberculosis FAS is not just a static scaffold for the catalytic domains, but may play an active role in coordinating fatty acid synthesis. The conception of M. tuberculosis FAS as a highly dynamic assembly of domains revises the view on bacterial type I fatty acid synthesis and might inspire new strategies for inhibition of de novo fatty acid synthesis in M. tuberculosis.
