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Hexameric assembly of the AAA+ protein McrB is necessary for GTPase activity

McrBC is one of the three modification-dependent restriction enzymes encoded by the Escherichia coli K12 chromosome. Amongst restriction enzymes, McrBC and its close homologues are unique in employing the AAA+ domain for GTP hydrolysis-dependent activation of DNA cleavage. The GTPase activity of Mcr...

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Detalles Bibliográficos
Autores principales: Nirwan, Neha, Singh, Pratima, Mishra, Gyana Gourab, Johnson, Christopher M, Szczelkun, Mark D, Inoue, Katsuaki, Vinothkumar, Kutti R, Saikrishnan, Kayarat
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344862/
https://www.ncbi.nlm.nih.gov/pubmed/30521042
http://dx.doi.org/10.1093/nar/gky1170
Descripción
Sumario:McrBC is one of the three modification-dependent restriction enzymes encoded by the Escherichia coli K12 chromosome. Amongst restriction enzymes, McrBC and its close homologues are unique in employing the AAA+ domain for GTP hydrolysis-dependent activation of DNA cleavage. The GTPase activity of McrB is stimulated by the endonuclease subunit McrC. It had been reported previously that McrB and McrC subunits oligomerise together into a high molecular weight species. Here we conclusively demonstrate using size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) and images obtained by electron cryomicroscopy that McrB exists as a hexamer in solution. Furthermore, based on SEC-MALS and SAXS analyses of McrBC and the structure of McrB, we propose that McrBC is a complex of two McrB hexamers bridged by two subunits of McrC, and that the complete assembly of this complex is integral to its enzymatic activity. We show that the nucleotide-dependent oligomerisation of McrB precedes GTP hydrolysis. Mutational studies show that, unlike other AAA+ proteins, the catalytic Walker B aspartate is required for oligomerisation.