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

Neha Nirwan, Praitma Singh, Gyana Gourab Mishra, Christopher M. Johnson, Mark Szczelkun, Katsuaki Inoue, Kutti R Vinothkumar, Kayarat Saikrishnan

Research output: Contribution to journalArticle (Academic Journal)peer-review

8 Citations (Scopus)
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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.
Original languageEnglish
Pages (from-to)868-882
Number of pages15
JournalNucleic Acids Research
Issue number2
Early online date6 Dec 2018
Publication statusPublished - 25 Jan 2019


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