Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Viruses utilize ATP-powered motors for essential processes like genome packaging.
• The phage lambda motor's ATP-binding motif (Walker-B) and conserved residues are crucial for its function.

Purpose of the Study:

• To confirm the Walker-B motif in the phage lambda motor.
• To elucidate the mechanistic roles of conserved residues in ATP binding, hydrolysis, and DNA translocation.

Main Methods:

• Genetic analysis of mutant phage lambda.
• Biochemical assays to measure ATPase activity.
• Single-molecule measurements of motor velocity and DNA translocation.
• Molecular dynamics simulations.

Main Results:

• Most mutations in conserved hydrophobic residues drastically reduced phage yield, but nine mutants retained partial activity.
• Mutations at residue D178, critical for ATP coordination, were lethal except for D178E, which slowed hydrolysis.
• Single-molecule data revealed specific effects of mutations on ATP binding, hydrolysis, and motor slipping.

Conclusions:

• Conserved residues in the phage lambda motor's Walker-B motif are essential for efficient ATP binding, hydrolysis, and DNA translocation.
• Specific mutations differentially affect motor steps, providing insights into the mechanism of ATP-coupled DNA packaging.
• The aspartate at D178 plays a key role in catalytic activity and nucleotide polarization.