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

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.

Fig. 1. Determination of the ATPase activity of gp16

(A) Example data showing how to determine an initial velocity from the burst of P_i as released from the ATPase reaction by gp16. The released P_i is scavenged by MDCC-PBP, which are excitated at 425 nm and the emission was detected at 464 nm by fluorometer. The initial velocity V_init (dashed line) was described as µM s⁻¹. (B) Optimization of the concentration of gp16 under 1 mM of ATP and 2 µM of MDCC-PBP. Each data points represent the initial velocity of the ATPase activity in the given concentration of gp16 that are expressed as linear scale.

Fig. 1. Determination of the ATPase activity of gp16

(A) Example data showing how to determine an initial velocity from the burst of P_i as released from the ATPase reaction by gp16. The released P_i is scavenged by MDCC-PBP, which are excitated at 425 nm and the emission was detected at 464 nm by fluorometer. The initial velocity V_init (dashed line) was described as µM s⁻¹. (B) Optimization of the concentration of gp16 under 1 mM of ATP and 2 µM of MDCC-PBP. Each data points represent the initial velocity of the ATPase activity in the given concentration of gp16 that are expressed as linear scale.

Fig. 2. Determination of kinetic parameters for an intrinsic ATPase activity of gp16

The rate of ATPase activity of gp16 (k_cat) was determined by linear regression plotting (Hanes-Woolf plot) based on the Michaelis-Menten equation. k_cat = 0.016 ± 0.002 s⁻¹, K_m = 351.0 ± 34.0 µM (n=7). Inset box shows the stimulation effect of DNA on the ATPase activity of gp16 depending on the chemistry and structure. Stimulated V_max of gp16 ATPase activity by single-stranded poly dT (closed circles) and poly dA (closed triangles) were compared to that by double-stranded 54 bp DNA (open squares) and pRNA (open diamonds). Open circles represent the ATPase activity of gp16 in the absence of DNA.

Fig. 3. Binding preference of gp16 to DNA observed by fluorescence single molecule imaging microscopy

(A) Fluorescence images of fluorescent DNA bound to gp16-immobilized surface. The range of either Cy5-poly dT or Cy3-poly dA was incubated inside gp16-immobilized microchamber. For the doubles-stranded poly dA:poly dT, Cy3-poly dA and Cy5-poly dT were hybridized to anneal each other and purified from 12% native PAGE. Phi29 Cy3-pRNA binding to gp16 was compared to the DNA bindings as the same manner. (B) Binding affinity comparison of gp16 between Cy5-poly dT (closed circles) and Cy3-poly dA (open circles) by plotting their mean fluorescence intensities over the concentrations of the fluorescent DNAs. The seleced area is 37 µm × 77 µm.

Fig. 3. Binding preference of gp16 to DNA observed by fluorescence single molecule imaging microscopy

(A) Fluorescence images of fluorescent DNA bound to gp16-immobilized surface. The range of either Cy5-poly dT or Cy3-poly dA was incubated inside gp16-immobilized microchamber. For the doubles-stranded poly dA:poly dT, Cy3-poly dA and Cy5-poly dT were hybridized to anneal each other and purified from 12% native PAGE. Phi29 Cy3-pRNA binding to gp16 was compared to the DNA bindings as the same manner. (B) Binding affinity comparison of gp16 between Cy5-poly dT (closed circles) and Cy3-poly dA (open circles) by plotting their mean fluorescence intensities over the concentrations of the fluorescent DNAs. The seleced area is 37 µm × 77 µm.