ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system

Abstract

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.

FIG. 1.

Purification of TrwK. Aliquots of pooled fractions corresponding to the different stages of the purification were resolved in sodium dodecyl sulfate polyacrylamide gels (10%) and stained with Coomassie blue. Lane a, cell lysates; lane b, SP-Sepharose flowthrough; lane c, Q-Sepharose elution; lane d, second Q-Sepharose elution; lane e, isocratic Superdex-200 elution. Both bands in lane e were excised manually from the gel and subjected to in-gel digestion with trypsin and analyzed by LC-MS/MS. Peptide fragment fingerprinting identified the larger band as TrwK and the smaller band as a TrwK N-terminal deletion (see the supplemental material).

FIG. 2.

TrwK ATP hydrolase activity. TrwK ATPase activity was monitored by the decrease of NADH absorbance at 340 nm (A) in the presence of 75 mM NaCl (trace b) or 75 mM KAc (trace c). The control is shown in trace a. The effects of salt concentrations (B) and pH (C) are represented.

FIG. 3.

Kinetic analysis of TrwK ATP hydrolysis. (A) ATP hydrolysis rates represented as a function of ATP concentration fit better with a Hill equation (solid line) than with a Michaelis-Menten equation (dashed line). (B) Hill plot for ATP hydrolysis by TrwK. The slope of the line for the low ATP concentrations (<1 mM) and medium ATP concentrations (>1 mM) were estimated as apparent Hill coefficients of 0.5 and 0.75, respectively.

FIG. 4.

Sedimentation velocity analysis of TrwK. The sedimentation velocity profiles and distribution of sedimentation coefficients were obtained from experiments conducted either in HEPES buffer at pH 7.25 (A, B) or PIPES buffer at pH 6.45 (C, D). The labeled peaks in panels B and D correspond to monomers (a), dimers (b), trimers (c), and hexamers (d). OD, optical density [c(s)].

FIG. 5.

Electron microscopy of TrwK oligomers. Protein eluted from a size exclusion chromatography column (GL 10/30 Superdex200) at an MW_app of ∼500 (indicated by the asterisk in panel A; insert, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of fractions from the chromatogram) were stained with uranyl acetate and analyzed by electron microscopy (B; scale bar, 50 nm). Particles (4,907) were selected, aligned, and classified by maximum likelihood multireference alignment methods. Three of the class averages (200 to 300 particles) are represented in panel C (scale bar, 5 nm).

FIG. 6.

A model showing the three hexameric ATPases in conjugative T4SS. T4SS in conjugative bacteria contain three hexameric ATPases on the cytoplasmic side of the inner membrane. According to our model, TrwK (VirB4) will be anchored to the membrane by interactions with TrwM (VirB3) and will be involved in energizing substrate transport. TrwK oligomerization will also promote pilus assembly and stabilization of the core components, TrwG and TrwE (VirB8 and VirB10, respectively). TrwD (VirB11) might interact directly with the membrane and/or with TrwK (VirB4) and will provide energy to unfold protein substrates so they can be transported through the membrane. TrwB (VirD4) will be directly attached to the membrane by its transmembrane region at its N-terminal domain and will provide the energy to pump DNA. OM, outer membrane; IM, inner membrane.