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

Abstract

The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 Å structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to α₃β₃ of F₁-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5'-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.

Fig. 1

Architecture of McrB∆NC complex. a Domain organization of McrB and McrC. b Structure of McrB∆NC and c of an McrB∆N protomer. The bound nucleotide is shown in stick representation and the magnesium ion as a green sphere. d Architecture of McrB∆N hexamer. The buried surface area at the interfaces are mentioned. Cryo-electron microscopic densities shown as isosurface mesh at 1.5 σ for e GNP-Mg²⁺ at the AB and f GDP at the DE interface. g Interactions with the guanine base at the AB interface that establish specificity for guanine. Green dashed lines represent potential hydrogen bonds

Fig. 2

Nucleotide binding at the McrBΔN interfaces. a–f Six panels, arranged in a clockwise cyclic manner, illustrating the interactions made with the nucleotide at the six interfaces. Green dotted lines represent potential hydrogen bonds (<3.6 Å) and ionic interactions (<4 Å). g Size exclusion chromatographic profile of McrB and its mutants in the presence of GTP using Superdex 200 column and in the presence of McrC and GTP using Superose 6 column (inset). h Comparison of GNP binding by McrB and its mutants. The binding curves of McrB^R348A and McrB^R349A overlapped. i Comparison of the GTPase activity of McrB and its mutants in the presence of McrC. j Nucleolytic activity of McrB and its mutants in the presence of McrC

Fig. 3

Remodeling of McrBΔN by McrC. a The dimer of McrC (ribbon representation) in complex with McrB∆N hexamer (surface representation). Note that this view looks at the face of the ring opposite to that in Fig. 1d. b The structure of an McrC monomer with the nuclease catalytic residues highlighted. c The asymmetric interaction of a monomer of McrC with McrB∆N hexamer. For clarity, protomer E of McrB∆N has been hidden. d Structure of McrB∆NC with the pore of the ring blocked by McrC is shown in the center. Around this figure, the interactions between McrC and the six McrB∆N protomers are illustrated. The inset is a zoom of the interactions between the helix bundle of McrC and L3 and C-terminal domain of McrB∆N protomers, highlighting the McrC-mediated remodeling of L3 in subunits A, B, C and D. The side chains of L3 residues are colored differently. Note the change in position of McrB-V341 (turquoise) and McrB-L339 (green), marked in dashed circles. The steric interaction between the helix bundle of McrC and L3 of B and C subunits are indicated by arrows (gray)

Fig. 4

Sequential GTP hydrolysis by McrBC. a–f A zoom of loop L3 of McrB∆N protomers highlighting the conformational changes in them and the interaction network with the catalytically important McrB-Glu280 and Asn333. Overlaid on each panel is the faded image of this region in protomer C. The panels are arranged to follow the clockwise cyclic arrangement of the protomers in the hexamer. g A schematic model illustrating the McrC (yellow) stimulated GTP hydrolysis by McrB. For clarity, only one hexamer and an McrC monomer are shown, and the ring is viewed from the McrC side as in Fig. 3a. GTP* represents the transition state. The pointed end of McrC is located at the interface of GDP-to-GTP exchange and the rounded end is at the interface of GTP hydrolysis