title: Dual nucleotide specificity determinants of an infection aborting anticodon nuclease.
abstract: The anticodon nuclease (ACNase) PrrC is silenced by a DNA restriction-modification (RM) protein and activated by a phage T4-encoded restriction inhibitor. The activation is driven by GTP hydrolysis while dTTP, which accumulates during the infection, stabilizes the active form. We show here, first, that the ABC-ATPase N-domains of PrrC can accommodate the two nucleotides simultaneously. Second, mutating a sequence motif that distinguishes the N-domain of PrrC from typical ABC-ATPases implicates three residues in the specificity for dTTP. Third, failure to bind dTTP or its deprivation hastened the centrifugal sedimentation of PrrC, possibly due to exposed sticky PrrC surfaces. Fourth, dTTP inhibited the GTPase activity of PrrC, probably by preventing GDP from leaving. These observations, correlated with relevant traits of a related ACNase, further suggest that PrrC utilizes GTP at canonical ABC-ATPase sites and binds dTTP to distinct sites exposed upon disruption of the ACNase-silencing interaction with the RM partner.
authors.affiliation: Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Krutkina
authors.affiliation: Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel.
authors.forename: Daniel
authors.initials: D
authors.lastname: Klaiman
authors.affiliation: Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel.
authors.forename: Tamar
authors.initials: T
authors.lastname: Margalit
authors.affiliation: NanoTemper, Flössergasse 4, 81369 München, Germany.
authors.forename: Moran
authors.initials: M
authors.lastname: Jerabeck-Willemsen
authors.affiliation: Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel. Electronic address: gabika@tauex.tau.ac.il.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Kaufmann
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics*; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D009711:Nucleotides; C084965:PrrC protein, E coli; D012260:Ribonucleases; C049345:anticodon nuclease; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases
keywords: ABC-ATPase; Anticodon nuclease; DNA restriction and anti-restriction; DTTP binding; Phage T4-exclusion; PrrC