The GTP-binding domain of McrB: more than just a variation on a common theme?
The methylation-dependent restriction endonuclease McrBC from Escherichia coli K12 cleaves DNA containing two R(m)C dinucleotides separated by about 40 to 2000 base-pairs. McrBC is unique in that cleavage is totally dependent on GTP hydrolysis. McrB is the GTP binding and hydrolyzing subunit, whereas MrC stimulates its GTP hydrolysis. The C-terminal part of McrB contains 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-binding domain of McrB. Amino acid substitutions were initially performed in the three proposed GTP-binding motifs. Whereas substitutions in motif 1 (P203V) 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 nucleotide specificity from GTP to XTP, but results in a lack of GTPase stimulation by McrC. The finding that McrB is not a typical G protein motivated us to perform a search for similar sequences in DNA databases. Eight microbial sequences were found, mainly from unfinished sequencing projects, with highly conserved sequence blocks within a presumptive GTP-binding domain. From the five sequences showing the highest homology, 17 invariant charged or polar residues outside the classical three GTP-binding motifs were identified and subsequently exchanged to alanine. Several mutations specifically affect GTP affinity and/or GTPase activity. Our data allow us to conclude that 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 function.
Pieper
U
U
Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany. Uwe.Pieper@chemie.bio.uni-giessen.de
Schweitzer
T
T
Groll
D H
DH
Gast
F U
FU
Pingoud
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D020558:GTP Phosphohydrolases; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D009711:Nucleotides; D006160:Guanosine Triphosphate; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease; D020558:GTP Phosphohydrolases