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. 2013 Aug 15;443(1):20-7.
doi: 10.1016/j.virol.2013.04.004. Epub 2013 May 22.

The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily

Chad Schwartz  1 , Gian Marco De Donatis, Huaming Fang, Peixuan Guo
Affiliations

The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily

Chad Schwartz et al. Virology. .

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).

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Figures

Fig. 1
Fig. 1. Depiction of the phi29 DNA packaging motor structure and function
A schematic of hexameric pRNA (left, top) and AFM images of loop-extended hexameric pRNA (top, right) (Shu et al., in press). Illustrations of the phi29 DNA packaging motor and a pRNA hexamer: side view (bottom, left) and bottom view (bottom, right).
Fig. 2
Fig. 2
(A) 6% Native PAGE using a non-denaturing detergent which fractionates by size reveals distinct bands characteristic of six oligomeric states; the top, hexameric band increased as the concentration of protein is increased (15 μM, 17.5 μM, 20 μM). Oligomeric states were assigned based on the mobility of marker proteins in the Native PAGE Mark kit. (B) EMSA of native eGFP-gp16 (3 μM) with short, 40 bp Cy3-dsDNA (300 nM) and ATP (30 mM) or γ-S-ATP (1.25 mM). The GFP channel (left) shows migration of the ATPase, whereas the Cy3 channel (right) indicates the migration of the dsDNA. Two distinct states of ATPase exist after addition of ATP to the gp16:DNA complex.
Fig. 3
Fig. 3. ATPase gp16 binds to DNA in a 6:1 molar ratio
EMSA of 3 μM gp16 and dsDNA (A) where free dsDNA band disappears (bottom right) as the molar ratio of gp16: dsDNA reaches 6:1. (B) Capillary electrophoresis of eGFP-gp16 and Cy3-DNA complexes after quantification of fluorescent peaks. Data are plotted as a ratio of total DNA versus bound DNA and plateaus at 0.5 μM, a concentration six times less than the fixed molar concentration of ATPase gp16. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4. ATPase gp16 contains a Walker A and Walker B motif typical of the AAA+ family
Assay of gp16 ATPase activity was described previously (Lee et al., 2008) (A). Walker A G27D and Walker B D118E/E119D mutants of gp16 prevent ATP hydrolysis. Capillary electrophoresis quantification of dsDNA binding to mutant and wildtype gp16 (B). Walker B D118E/E119D mutant retains binding capability to dsDNA despite addition of ATP. EMSA of mutant and wildtype ATPase (C). DNA binding is diminished with the Walker A G27D mutant but is retained in the Walker B D118E/E119D mutant with addition of ATP or γ-S-ATP. The results were comparable with Walker B E119A mutant. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Viral assembly inhibition assay using a binomial distribution revealing that gp16 is a hexamer in the DNA packaging motor (Trottier and Guo, 1997). Theoretical plot of percent Walker B mutant gp16 versus yield of infectious virions in in vitro phage assembly assays. Predictions were made with the equation (p+q)Z=(Z0)pZ+(Z1)pZ-1q+(Z2)pZ-2q2+⋯+(ZZ-1)pqZ-1+(ZZ)qZ=∑M=0Z(ZM)pZ-MqM, where p is the percentage of wild-type eGFP-gp16; q is the percentage of eGFP-gp16/ED; Z, is the total number of eGFP-gp16 per procapsid or gp3-DNA; M is the number of mutant eGFP-gp16 in the phi29 DNA packaging motor; and p+q = 1 (Trottier and Guo, 1997).
Fig. 6
Fig. 6. Hexameric push through a one-way valve mechanism
(Schwartz et al., 2012). A conformational change in the hexameric ATPase occurs subsequently after binding to ATP which confers an increase in binding affinity to dsDNA. Release of inorganic phosphate from the ATPase complex results in a power stroke to push the genomic dsDNA through the one-way valve of the connector portal protein into the capsid shell.
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