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

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.

Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes

“…Negative-stain EM indicates that the Asp356Ala mutation has a higher propensity to disrupt the TgMcrB AAA hexamer than the Glu357Ala mutation (Figure 2e), consistent with their distinct functions in nucleotide binding/stabilization versus catalysis. This result mirrors the different effects on oligomerization observed when the corresponding residues (Asp279 and Glu280) were mutated in EcMcrB (Nirwan et al, 2019a). Notably, the conserved McrB consensus loop ( 409 MNxxDR 414 ) replaces Sensor I and is located close to the γ-phosphate (Figure 2 and Supplementary Data S1).…”

“…Previous studies reported that EcMcrBC complexes form tetradecameric assemblies in vitro (Nirwan et al, 2019a; Panne et al, 2001). In our hands, dimeric McrBC complexes generated using the full-length Tg and Ec proteins exhibit a high degree of conformational variability, which prevented us from calculating interpretable maps for these larger oligomeric states and limited our ability to analyze the dimer interface between the two McrC subunits.…”

“…McrBC is a two-component MDRS that in E. coli (Ec) restricts phage DNA and foreign DNA containing methylated cytosines (Luria and Human, 1952; Weigele and Raleigh, 2016). EcMcrB consists of an N-terminal DNA-binding domain that targets fully or hemi-methylated R M C sites (where R is a purine base and M C is a 4-methyl-, 5-methyl- or 5-hydroxymethyl-cytosine) (Gast et al, 1997; Kruger et al, 1995; Pieper et al, 1999b; Sukackaite et al, 2012; Sutherland et al, 1992; Zagorskaite et al, 2018) and a C-terminal AAA+ (extended A TPases Associated with various cellular Activities) domain that hydrolyzes GTP and oligomerizes into hexamers (Nirwan et al, 2019a; Panne et al, 2001). EcMcrB’s basal GTPase activity (~0.5-1 min −1 ) is stimulated ~30-40-fold in vitro via interaction with its partner EcMcrC (Pieper et al, 1999b), a PD-(D/E)xK family endonuclease that cannot stably bind DNA on its own and thus associates with the hexameric McrB AAA+ ring (Panne et al, 2001).…”

Structure-based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC

“…McrB is unique among AAA+ proteins as it hydrolyzes GTP rather than ATP 1 . In the presence of GTP, monomeric McrB oligomerize to hexamers 13 . Furthermore, upon binding of the endonuclease McrC, a higher oligomeric form—tetradecamers of 12 McrB and 2 McrC protomers—is formed 13 .…”

“…In the presence of GTP, monomeric McrB oligomerize to hexamers 13 . Furthermore, upon binding of the endonuclease McrC, a higher oligomeric form—tetradecamers of 12 McrB and 2 McrC protomers—is formed 13 . The McrB GTPase activity is very low and is stimulated ~30-fold by McrC 3,13 .…”