Defining the ATPase center of bacteriophage T4 DNA packaging machine: requirement for a catalytic glutamate residue in the large terminase protein gp17.
Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT(161-167)) and Walker B (MIYID(251-255)) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine.
Goetzinger
Katherine R
KR
Department of Biology, The Catholic University of America, 103 McCort Ward Hall, 620 Michigan Ave, NE Washington, DC 20064, USA.
Rao
Venigalla B
VB
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017122:Bacteriophage T4 / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000502:physiology; D004926:Escherichia coli / Q000821:virology; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019065:Virus Assembly / Q000235:genetics*
D004279:DNA, Viral; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4; D000251:Adenosine Triphosphatases