Abstract
McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC complexes use different DNA-binding domains, these contribute to the same general GTP-recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis cycle. Our data provide insights into the conserved molecular mechanisms governing McrB family AAA + motors.
Highlights
McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA
The cryo-EM map reveals that TgMcrBAAA forms a ring-shaped, homohexameric assembly with six nucleotides bound at the subunit interfaces, similar to the closed-ring assembly seen in type I AAA ATPases[42,43]
Our structural analysis reveals that TgMcrBAAA forms an asymmetric hexamer, similar to the architecture adopted by many other AAA+ family proteins[58,59,60,61,62,63,64,65]
Summary
McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The slow progress in developing new drugs to combat these emerging “superbugs” and the rapid exchange of resistance genes among microbial populations has intensified the need for alternative therapeutic strategies[3] One such strategy employs bacteriophages (phages)—viruses that infect a bacterial host, replicate, and induce cell lysis to release the mature phage progeny, killing the host in the process[4]. One significant hurdle is that bacteria have evolved an array of defense mechanisms, including restriction modification systems, modificationdependent restrictions systems (MDRSs), phage-exclusion systems, and CRISPR-Cas adaptive immune systems, that can hinder phage infection and diminish their subsequent killing potential[8,9] These machineries lack eukaryotic homologs and are conserved across antibiotic-resistant bacteria like methicillin-resistant Staphylococcus aureus, Clostridium difficile, and Klebsiella pneumoniae, making their components promising candidates for targeted inhibition. EcMcrB consists of an N-terminal DNA-binding domain that targets fully or hemi-methylated RMC sites (where R is a purine base and MC is a 4-methyl-, 5-methyl-, or 5-hydroxymethyl-cytosine)[14,15,16,17,18,19], and a C-terminal AAA+
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