title: Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging.
abstract: During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectable levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectable yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type (WT) but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A and K76R, while G81A and S83A exhibit strong impairments, consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest that these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.
authors.affiliation: Department of Physics, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Damian
authors.initials: D
authors.lastname: delToro
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Ortiz
authors.affiliation: Department of Physics, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Mariam
authors.initials: M
authors.lastname: Ordyan
authors.affiliation: Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Sippy
authors.affiliation: Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Choon-Seok
authors.initials: CS
authors.lastname: Oh
authors.affiliation: Department of Physics, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Keller
authors.affiliation: Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. Electronic address: michael-feiss@uiowa.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Feiss
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA. Electronic address: carlos.catalano@ucdenver.edu.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Catalano
authors.affiliation: Department of Physics, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: des@ucsd.edu.
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Smith
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D042003:DNA Packaging / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D006868:Hydrolysis; D008958:Models, Molecular; D017354:Point Mutation / Q000235:genetics; D000072417:Protein Domains / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: ATPase; Optical tweezers; mutagenesis of Walker-A motif; terminase; viral DNA packaging motor