Article

Strand and nucleotide-dependent ATPase activity of gp16 of bacterial virus phi29 DNA packaging motor

Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
Virology (Impact Factor: 3.32). 09/2008; 380(1):69-74. DOI: 10.1016/j.virol.2008.07.003
Source: PubMed

ABSTRACT

Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5'/3' helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k(cat) and K(m) in the absence of DNA was 0.016 s(-1) and 351.0 microM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine)>dsDNA>poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.

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    • "The motor undergoes a cycle of conformational changes between two distinct states during its interaction with ATP; the initial step is the binding of ATP that results in a reduction of entropy in the ATPase by a conformational change (Guo et al., 1987c; Ibarra et al., 2001; Lee et al., 2008). The entropy lost is compensated by a subsequent step of ATP hydrolysis resulting in entropy increase with another conformational change. "
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    ABSTRACT: Biomotors have been classified into linear and rotational motors. For 35 years, it has been popularly believed that viral dsDNA-packaging apparatuses are pentameric rotation motors. Recently, a third class of hexameric motor has been found in bacteriophage phi29 that utilizes a mechanism of revolution without rotation, friction, coiling, or torque. This review addresses how packaging motors control dsDNA one-way traffic; how four electropositive layers in the channel interact with the electronegative phosphate backbone to generate four steps in translocating one dsDNA helix; how motors resolve the mismatch between 10.5 bases and 12 connector subunits per cycle of revolution; and how ATP regulates sequential action of motor ATPase. Since motors with all number of subunits can utilize the revolution mechanism, this finding helps resolve puzzles and debates concerning the oligomeric nature of packaging motors in many phage systems. This revolution mechanism helps to solve the undesirable dsDNA supercoiling issue involved in rotation.
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    • "Enzymatic activity via fluorescent labeling was described previously (Lee et al., 2008). Briefly, a phosphate binding protein conjugated to a fluorescent probe that senses the binding of phosphate was used to assay ATP hydrolysis. "
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    ABSTRACT: The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue).
    Full-text · Article · May 2013 · Virology
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    • "The combination of gp16 and gp3-DNA, while only marginally more effective at ATP hydrolysis than gp16 and pRNA, appears less active than gp16 by itself indicating a potential regulatory effect of force yielding ATPase activity, which may be seen in conjunction with gp3-DNA, as opposed to indiscriminate bulk ATPase activity. Though gp3 is not required for binding of DNA to gp16 (Koti et al., 2008), the presented data indicate that gp3-DNA increases the ATPase activity of gp16 above that of isolated DNA (Lee et al., 2008) consistent with previous reports that it increases genomic packaging efficiency by several orders of magnitude (Koti et al., 2008). When gp16 and gp10 (connector) were introduced into the reaction, the catalytic rate constant of ATP hydrolysis was 0.322 s À 1 and the Michaelis constant was 109 mM, and it is noteworthy that these kinetics resemble the pRNA-gp16 kinetics (see Fig. 3). "
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    ABSTRACT: Presented is a detailed kinetic evaluation of the motor component interactions of the DNA translocation ATPase of Bacillus subtilis bacteriophage φ29. The components of the φ29 DNA packaging motor, comprised of both protein and non-protein parts, act in a coordinated manner to translocate DNA into a viral capsid, despite entropically unfavorable conditions. The precise nature of this coordination remains under investigation but recent results have shown that the gp16 pentamer acts to propel the genomic DNA in 10 base pair bursts, implying inter-subunit synchronization. We observe an emergent tandem coordination behavior in the ATPase activity of gp16 as demonstrated by a Hill coefficient of 2.4±0.2, as differentiated from its activity in DNA packaging which has been shown to have a unity Hill coefficient. Due to its relative strength and DNA packaging efficiency, understanding the molecular mechanism of force generation may prove useful to various nanotechnology applications including gene therapy, control of biological ATPases, and the powering of nanoscale mechanical devices.
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