Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif.
Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107
-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+
•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor.
delToro, D; Ortiz, D; Ordyan, M; Pajak, J; Sippy, J; Catala, A; Oh, C-S; Vu, A; Arya, G; Smith, DE; Catalano, CE; Feiss, M
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