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

Nucleic Acids Res. 2019 Jan 25;47(2):868-882. doi: 10.1093/nar/gky1170.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy
  • DNA Restriction Enzymes / genetics
  • DNA Restriction Enzymes / metabolism*
  • DNA Restriction Enzymes / ultrastructure
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Escherichia coli Proteins / ultrastructure
  • GTP Phosphohydrolases / metabolism*
  • Guanosine Triphosphate / metabolism
  • Mutation
  • Protein Multimerization
  • Scattering, Small Angle
  • X-Ray Diffraction

Substances

  • Escherichia coli Proteins
  • mcrC protein, E coli
  • Guanosine Triphosphate
  • DNA Restriction Enzymes
  • mcrB protein, E coli
  • GTP Phosphohydrolases