The GTP-binding domain of McrB: More than just a variation on a common theme?

The methylation-dependent restriction endonuclease McrBC from Escherichia c oli K12 cleaves DNA containing two (RC)-C-m dinucleotides separated by abou t 40 to 2000 base-pairs. McrBC is unique in that cleavage is totally depend ent on GTP hydrolysis. McrB is the GTP binding and hydrolyzing subunit, whe reas MrC stimulates its GTP hydrolysis. The C-terminal part of McrB contain s the sequences characteristic for GTP-binding proteins, consisting of the GxxxxGK(S/T) motif (position 201-208), followed by the DxxG motif (position 300-303). The third motif (NKxD) is present only in a non-canonical form ( NTAD 333-336). Here we report a mutational analysis of the putative GTP-bin ding domain of McrB. Amino acid substitutions were initially performed in t he three proposed GTP-binding motifs. Whereas substitutions in motif 1 (P20 3V) and 2 (D300N) show the expected, albeit modest effects, mutation in the motif 3 is at variance with the expectations. Unlike the corresponding EF Tu and ras-p21 variants, the D336N mutation in McrB does not change the nuc leotide specificity from GTP to XTP, but results in a lack of GTPase stimul ation by McrC. The finding that McrB is not a typical G protein motivated u s to perform a search for similar sequences in DNA databases. Eight microbi al sequences were found, mainly from unfinished sequencing projects, with h ighly conserved sequence blocks within a presumptive GTP-binding domain. Fr om the five sequences showing the highest homology, 17 invariant charged or polar residues outside the classical three GTP-binding motifs were identif ied and subsequently exchanged to alanine. Several mutations specifically a ffect GTP affinity and/or GTPase activity. Our data allow us to conclude th at McrB is not a typical member of the superfamily of GTP-binding proteins, but defines a new subfamily within the superfamily of GTP-binding proteins , together with similar prokaryotic proteins of as yet unidentified functio n. (C) 1999 Academic Press.