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

Abstract

13McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving 14 foreign DNA. The GTP-specific AAA+ protein McrB powers translocation along DNA and its 15 hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM 16 structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB 17 hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are 18 intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active 19 site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the 20 asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC 21 complexes use different DNA-binding domains, these contribute to the same general GTP-22 recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-23 subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis 24 cycle. Our data provide novel insights into the conserved molecular mechanisms governing 25 McrB family AAA+ motors. upon research conducted at NE-CAT beamlines (24-ID-C and 24-ID-E) under the general user