title: Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function.
abstract: The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH(-) nucleophile. The ATPase Walker B motif, thus, may be redefined as "β-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid".
authors.affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue NE, Washington, DC, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Kondabagil
authors.affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue NE, Washington, DC, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Dai
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors.forename: Reza
authors.initials: R
authors.lastname: Vafabakhsh
authors.affiliation: Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Howard Hughes Medical Institute, Urbana, IL, USA.
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
authors.affiliation: Department of Biology, St. Andrews University, NC, USA.
authors.forename: Bonnie
authors.initials: B
authors.lastname: Draper
authors.affiliation: Department of Biology, The Catholic University of America, 620 Michigan Avenue NE, Washington, DC, USA. Electronic address: rao@cua.edu.
authors.forename: Venigalla B
authors.initials: VB
authors.lastname: Rao
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D042003:DNA Packaging / Q000235:genetics / Q000502:physiology*; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D005786:Gene Expression Regulation; D015723:Gene Library; D009154:Mutation; D011487:Protein Conformation
chemical_list: D004279:DNA, Viral; D000251:Adenosine Triphosphatases
keywords: ATPase; Bacteriophage T4; DNA packaging; Molecular motor; Virus assembly; Walker B motif