ATPase center of bacteriophage λ terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids

• Published in: Journal of molecular biology 2000-09, Vol.302 (4), p.777-795
• Main Authors: Hang, Julie Qi, Tack, Brian F., Feiss, Michael
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acid Substitution - genetics; Attachment Sites, Microbiological - genetics; Bacteriophage lambda - enzymology; Bacteriophage lambda - genetics; Bacteriophage lambda - physiology; Base Sequence; Binding Sites; Chromatography, High Pressure Liquid; cohesive ends; DNA packaging; DNA, Viral - genetics; DNA, Viral - metabolism; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; endonuclease; helicase; Holoenzymes - chemistry; Holoenzymes - genetics; Holoenzymes - metabolism; Hydrolysis; Kinetics; Mutation - genetics; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Phage ^l; Photoaffinity Labels; Protein Footprinting; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Regulatory Sequences, Nucleic Acid - genetics; terminase; Trypsin - metabolism; Ultraviolet Rays; Virion - enzymology; Virion - genetics; Virion - physiology; Virus Assembly; virus DNA packaging; virus genome processing
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2000.4086

Terminase is the enzyme that mediates λ DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity ( K m=5 μM). To directly identify gpA’s ATP-interacting amino acids, holoterminase bearing a His 6-tag at the C terminus of gpA was UV-crosslinked with 8-N 3-[α- 32P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E 43SA Y 46QEGR 50, or the lysine of the second peptide, V 80GYS K 84MLL 87, indicating that Y 46 and K 84 were the 8-N 3-ATP-modified amino acids. To investigate their roles in λ DNA packaging, Y 46 was changed to E, A, and F, and K 84 was changed to E and A. Purified His 6-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y 46 and K 84 are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post- cos cleavage stages of λ DNA packaging.