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Publication - Professor Mark Szczelkun

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


    Nirwan, N, Singh, P, Mishra, GG, Johnson, CM, Szczelkun, M, Inoue, K, Vinothkumar, KR & Saikrishnan, K, 2019, ‘Hexameric assembly of the AAA+ protein McrB is necessary for GTPase activity’. Nucleic Acids Research, vol 47., pp. 868-882


    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.

    Full details in the University publications repository