title: The glutamate switch of bacteriophage T7 DNA helicase: role in coupling nucleotide triphosphate (NTP) and DNA binding to NTP hydrolysis.
abstract: The DNA helicase encoded by gene 4 of bacteriophage T7 forms a hexameric ring in the presence of dTTP, allowing it to bind DNA in its central core. The oligomerization also creates nucleotide-binding sites located at the interfaces of the subunits. DNA binding stimulates the hydrolysis of dTTP but the mechanism for this two-step control is not clear. We have identified a glutamate switch, analogous to the glutamate switch found in AAA+ enzymes that couples dTTP hydrolysis to DNA binding. A crystal structure of T7 helicase shows that a glutamate residue (Glu-343), located at the subunit interface, is positioned to catalyze a nucleophilic attack on the γ-phosphate of a bound nucleoside 5'-triphosphate. However, in the absence of a nucleotide, Glu-343 changes orientation, interacting with Arg-493 on the adjacent subunit. This interaction interrupts the interaction of Arg-493 with Asn-468 of the central β-hairpin, which in turn disrupts DNA binding. When Glu-343 is replaced with glutamine the altered helicase, unlike the wild-type helicase, binds DNA in the presence of dTDP. When both Arg-493 and Asn-468 are replaced with alanine, dTTP hydrolysis is no longer stimulated in the presence of DNA. Taken together, these results suggest that the orientation of Glu-343 plays a key role in coupling nucleotide hydrolysis to the binding of DNA.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Ajit K
authors.initials: AK
authors.lastname: Satapathy
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D020125:Mutation, Missense; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D013942:Thymine Nucleotides; D014764:Viral Proteins; D018698:Glutamic Acid; D019915:DNA Primase; C024157:thymidine 5'-triphosphate