Abstract
Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.
Highlights
Numerous DNA packaging models have been proposed, yet the basic mechanism is still a mystery [5,6,7]
Cryoelectron microscopy imaging and atomic structure of phage Phi29 portal are consistent with the symmetry mismatch model, which postulates that the mismatch between the 5-fold viral capsid and 12-fold portal allows an ATP-driven portal rotation that is coupled to DNA translocation [8, 9]
Two Walker B motifs have been proposed in gp17: 251MIYID255 [10, 19] and 463GVSVAKSLYMD473 [21, 22]
Summary
Numerous DNA packaging models have been proposed, yet the basic mechanism is still a mystery [5,6,7]. Biochemical analyses using a series of purified mutant proteins revealed that the mutants exhibited defects in ATP binding and lost the gp16-stimulated ATPase and in vitro DNA packaging activities These data define the N-terminal ATPase catalytic center in gp. This study reports the most thorough molecular genetic analysis of the Walker B motif from any ATPase motor, generating the first set of conditionally lethal mutants Implications of these results for the structure and function of ATPase motors in general are discussed
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