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. 2013 Mar 1;450(2):417-25.
doi: 10.1042/BJ20121599.

Structure and mechanism of the PilF DNA transformation ATPase from Thermus thermophilus

Richard F Collins  1 , Darin Hassan, Vijaykumar Karuppiah, Angela Thistlethwaite, Jeremy P Derrick
Affiliations

Structure and mechanism of the PilF DNA transformation ATPase from Thermus thermophilus

Richard F Collins et al. Biochem J. .

Abstract

Many Gram-negative bacteria contain specific systems for uptake of foreign DNA, which play a critical role in the acquisition of antibiotic resistance. The TtPilF (PilF ATPase from Thermus thermophilus) is required for high transformation efficiency, but its mechanism of action is unknown. In the present study, we show that TtPilF is able to bind to both DNA and RNA. The structure of TtPilF was determined by cryoelectron microscopy in the presence and absence of the ATP analogue p[NH]ppA (adenosine 5'-[β,γ-imido]triphosphate), at 10 and 12 Å (1 Å=0.1 nm) resolutions respectively. It consists of two distinct N- and C-terminal regions, separated by a short stem-like structure. Binding of p[NH]ppA induces structural changes in the C-terminal domains, which are transmitted via the stem to the N-terminal domains. Molecular models were generated for the apoenzyme and p[NH]ppA-bound states in the C-terminal regions by docking of a model based on a crystal structure from a closely related enzyme. Analysis of DNA binding by electron microscopy, using gold labelling, localized the binding site to the N-terminal domains. The results suggest a model in which DNA uptake by TtPilF is powered by ATP hydrolysis, causing conformational changes in the C-terminal domains, which are transmitted via the stem to take up DNA into the cell.

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Figures

Figure 1
Figure 1. Arrangement of domains within selected secretory ATPases
The figure compares the proposed domain arrangement in TtPilF (top) with EpsE from Vibrio cholerae (VcEpsE) [22] and GspE from A. fulgidus (AfGspE) [18].
Figure 2
Figure 2. Nucleic acid contamination of purified TtPilF
(A) UV absorption spectrum of TtPilF (continuous line) and BSA (broken line), both at 0.1 mg/ml, recorded in 25 mM Tris/HCl (pH 8.0), 100 mM NaCl and 10 mM MgCl2. (B) Agarose gel (1.5%) electrophoresis of TtPilF. A total of 50 μg of purified TtPilF was loaded in each lane. Lane 1 has TtPilF without pretreatment, lane 2 has TtPilF after treatment with 2 units of DNase (RNase-free) and lane 3 has TtPilF after treatment with 2 units of RNase (DNase-free). DNA markers are indicated to the left of the gel.
Figure 3
Figure 3. Binding of DNA to TtPilF
(A) Agarose gel (2%) band-shift assay: top panel, 3.85 μg of polyA; bottom panel, 0.2 μg of polyAT. TtPilF quantities used were: lane 1, 50 μg; lane 2, 0 μg; lane 3, 25 μg; lane 4, 50 μg; lane 5, 75 μg and lane 6, 100 μg. (B) Band-shift assay for TtPilF with 4 mM p[NH]ppA. TtPilF and polyA/polyAT quantities used were the same as in (A). (C) Comparison of DNase digestion of polyAT in the presence (top panel) and absence (bottom panel) of TtPilF. Lane 1 has 50 μg of TtPilF, lane 2 has 200 ng of polyAT, lanes 3–7 have both 50 μg of TtPilF and 0.2 μg of polyAT with DNaase (Sigma) added as follows: lane 3, 2 units; lane 4, 6 units; lane 5, 18 units; lane 6, 54 units; and lane 7, 150 units. AMPPNP, p[NH]ppA.
Figure 4
Figure 4. TtPilF ATPase activity
Results shown are means±S.D. (n=3).
Figure 5
Figure 5. Determination of TtPilF structure by single particle averaging and cryoelectron microscopy
(A) A sample field of ‘raw’ TtPilF particles embedded in thin vitreous ice. The inset shows examples of projection averages determined from the raw data (note that contrast is inverted for analysis so that protein appears white). The sample data shown are from the TtPilF–p[NH]ppA complex. Scale bar=1000 Å. (B) Surface-rendered three-dimensional volume of the six-fold symmetric TtPilF–p[NH]ppA complex (orthogonal views) displayed at a sigma value to accommodate 600 kDa of protein mass. The slab view has the foremost 50% of volume removed to display protein density through the complex. The figure was prepared using Chimera [37].
Figure 6
Figure 6. Structural changes in TtPilF on binding of p[NH]ppA and modelling of the C-domains into cryoelectron density maps
(A) Comparison of TtPilF apoprotein and p[NH]ppA-bound structures. Left-hand panel, TtPilF apoenzyme, contoured at 4.9σ, in red; right-hand panel, as left-hand panel, but with the TtPilF–p[NH]ppA complex superimposed, contoured at 4.8σ, in green. The resolution of the TtPilF–p[NH]ppA complex was truncated at 12 Å resolution; sigma levels of both maps were selected such that the volumes were equal. The arrow in the right-hand panel shows the region of the connector stem that contracts on binding of p[NH]ppA. (B) Modelling of TtPilF C-domains into cryoelectron density maps. Top and side views of apoprotein (left-hand panel) and p[NH]ppA-bound (right-hand panel) models for the C-domains, superimposed on their respective electron density maps. One protomer in each hexamer is coloured differently to indicate subunit contacts. (C) Comparison of TtPilF C-domains in the apoprotein and p[NH]ppA-bound states. Superinposition of ribbon plots of TtPilF C-domain models for the apoprotein (red) and p[NH]ppA-bound (green) forms. Left-hand panel: a single subunit, viewed from the side; the N2 domain in the apoprotein is circled, with an arrow to indicate the lateral movement that occurs on binding of p[NH]ppA. Right-hand panel: top view of the assembled hexamers; the dimensions of the interior cavity are unchanged, but the apoprotein structure has a wider diameter, as a result of the movement of the N2 domain. AMPPNP, p[NH]ppA.
Figure 7
Figure 7. DNA binding to TtPilF
(A) A montage of TtPilF particles following incubation with biotin–DNA/avidin–gold. Box size=240 Å×240 Å. (B) Two-dimensional projection averages of negatively stained TtPilF incubated with biotin–DNA/avidin–gold. The locations of higher density peaks associated with gold particles are indicated by broken-lined circles. Scale bar=50 Å. (C) Three-dimensional alignment of the avidin–gold label within the TtPilF complex. The TtPilF volume is shown in purple surface render, contoured at a level to accommodate 600 kDa of mass. The positions of the gold densities at –4σ are indicated in gold surface render. The blue wireframe shows cross-correlated negative densities superimposed from a TtPilF volume with no gold label, contoured at –4σ and highlighting the specific electron density attributable to the gold particles. Scale bar=100 Å.
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References

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