A critical coiled coil motif in the small terminase, gp16, from bacteriophage T4: insights into DNA packaging initiation and assembly of packaging motor.
Double-stranded DNA packaging in bacteriophages is driven by one of the most powerful force-generating molecular motors reported to date. The phage T4 motor is composed of the small terminase protein, gpl6 (18kDa), the large terminase protein, gp17 (70kDa), and the dodecameric portal protein gp20 (61kDa). gp16, which exists as an oligomer in solution, is involved in the recognition of the viral DNA substrate, the very first step in the DNA packaging pathway, and stimulates the ATPase and packaging activities associated with gp17. Sequence analyses using COILS2 revealed the presence of coiled coil motifs (CCMs) in gp16. Sixteen T4-family and numerous phage small terminases show CCMs in the corresponding region of the protein, suggesting a common structural and functional theme. Biochemical properties such as reversible thermal denaturation and analytical gel filtration data suggest that the central CCM-1 is critical for oligomerization of gp16. Mutations in CCM-1 that change the hydrophobicity of key residues, or pH 6.0, destabilized coiled coil interactions, resulting in a loss of gp16 oligomerization. The gp16 oligomers are in a dynamic equilibrium with lower M(r) intermediate species and monomer. Monomeric gp16 is unable to stimulate gp17-ATPase, an activity essential for DNA packaging, while conversion back into oligomeric form restored the activity. These data for the first time defined a CCM that is critical for structure and function of the small terminase. We postulate a packaging model in which the gp16 CCM is implicated in the regulation of packaging initiation and assembly of a supramolecular DNA packaging machine on the viral concatemer.
Kondabagil
Kiran R
KR
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
Rao
Venigalla B
VB
D000251:Adenosine Triphosphatases / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D042003:DNA Packaging; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004706:Endodeoxyribonucleases / Q000737:chemistry; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019065:Virus Assembly
D004268:DNA-Binding Proteins; C065037:Gp16 protein, Bacteriophage T4; D020409:Molecular Motor Proteins; D014764:Viral Proteins; C065038:gp17 protein, Enterobacteria phage T4; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4; D000251:Adenosine Triphosphatases