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The Traffic ATPase PilF Interacts With the Inner Membrane Platform of the DNA Translocator and Type IV Pili From Thermus thermophilus

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The Traffic ATPase PilF Interacts With the Inner Membrane Platform of the DNA Translocator and Type IV Pili From Thermus thermophilus

Kerstin Kruse et al. FEBS Open Bio.

Abstract

A major driving force for the adaptation of bacteria to changing environments is the uptake of naked DNA from the environment by natural transformation, which allows the acquisition of new capabilities. Uptake of the high molecular weight DNA is mediated by a complex transport machinery that spans the entire cell periphery. This DNA translocator catalyzes the binding and splitting of double-stranded DNA and translocation of single-stranded DNA into the cytoplasm, where it is recombined with the chromosome. The thermophilic bacterium Thermus thermophilus exhibits the highest transformation frequencies reported and is a model system to analyze the structure and function of this macromolecular transport machinery. Transport activity is powered by the traffic ATPase PilF, a soluble protein that forms hexameric complexes. Here, we demonstrate that PilF physically binds to an inner membrane assembly platform of the DNA translocator, comprising PilMNO, via the ATP-binding protein PilM. Binding to PilMNO or PilMN stimulates the ATPase activity of PilF ~ 2-fold, whereas there is no stimulation when binding to PilM or PilN alone. A PilMK26A variant defective in ATP binding still binds PilF and, together with PilN, stimulates PilF-mediated ATPase activity. PilF is unique in having three conserved GSPII (general secretory pathway II) domains (A-C) at its N terminus. Deletion analyses revealed that none of the GSPII domains is essential for binding PilMN, but GSPIIC is essential for PilMN-mediated stimulation of ATP hydrolysis by PilF. Our data suggest that PilM is a coupling protein that physically and functionally connects the soluble motor ATPase PilF to the DNA translocator via the PilMNO assembly platform.

Keywords: ATPase; DNA transporter; PilF; natural competence; thermophile; type IV pili.

Figures

Figure 1
Figure 1
Purification of PilMNO, PilMN, PilM, and PilN. His6‐PilMNO‐Strep (lane 1), His6‐PilMN‐Strep (lane 2), His6‐PilM (lane 3), and PilN‐Strep (lane 4) were purified from Escherichia coli BL21 (DE3) cells as described in Experimental procedures. PilMNO, PilMN, and PilN were isolated from membranes, whereas PilM was isolated from soluble fractions. To separate His6‐PilM (44 kDa), PilN (23 kDa), and PilO‐Strep (22.5 kDa), 4–20% SDS/PAGE was performed (lane 1). All other protein preparations were separated by 14% SDS/PAGE (A). The identity of the proteins was confirmed by western blot using polyclonal antisera against PilM (B) and PilN (C) or using Strep‐Tactin HRP conjugate to detect PilO‐Strep (D). Dotted lines indicate excised lanes from different but comparable gels or blots.
Figure 2
Figure 2
Complex formation of His6‐PilMNO‐Strep, His6‐PilMN‐Strep, and His6‐PilM. Complex formation was analyzed by 5–20% clear‐native PAGE (A). His6‐PilMN‐Strep formed two different complexes indicated with arrows. Native gels were stained with InstantBlue (Expedeon, Cambridge, UK). The protein bands corresponding to the ~ 195 kDa (1) and ~ 80 kDa (2) His6‐PilMN‐Strep complexes were cut out from the gel, incubated in Sol‐buffer (2% (w/v) SDS, 60 mm Na2CO3, 0.67% (v/v) 2‐mercaptoethanol) and loaded onto a 14% SDS gel. The proteins were stained with Coomassie blue (B). The dotted lines indicate excised lanes from the same gels, respectively.
Figure 3
Figure 3
PilMNO, PilMN, and PilM pull down PilF. His6‐PilMNO‐Strep (A), His6‐PilMN‐Strep (B), Strep‐PilM (C), and PilN‐Strep (D) were bound to a Strep‐Tactin column and incubated with purified His6‐PilF hexamers. As a negative control, His6‐PilF was incubated on an empty Strep‐Tactin column (E). After washing with one column volume buffer for ten times, proteins were eluted. Flow‐through (FT) and fivefold concentrates of the tenth wash fraction (W) and eluate (E) were separated by SDS/PAGE (14% gel for A, 12% for B–E) and stained with Coomassie blue. The dotted line indicates an excised lane from the same gel.
Figure 4
Figure 4
PilMN and PilMNO complexes stimulate PilF ATPase activity. ATP hydrolysis activity of PilF was measured by incubating 66.7 pmol PilF hexamer with increasing amounts of PilMN (black bars), PilMNO (dark gray bars), PilM (light gray bars), or PilN (white bars). Release of phosphate was measured over 15 min. Shown are means ± empirical standard deviations of at least three independent measurements.
Figure 5
Figure 5
PilMK26A does not bind ATP but stimulates PilF ATPase activity. Two micrograms purified His6‐PilM and His‐PilMK26A was separated by SDS/PAGE. The dotted line indicates an excised lane from the same gel (A). ATP binding by PilM variants was analyzed by incubating 2 μg of purified protein with [α‐32P]‐ATP and photo cross‐linking. Proteins were precipitated and separated by SDS/PAGE. Radioactivity was detected on a storage phosphor screen (B). To analyze ATP content after purification, proteins were precipitated with TCA on ice. Na‐TES buffer and K2CO3 were added to adjust pH. After centrifugation, the ATP content of the supernatant was determined using firefly lantern extract as described in Experimental procedures (C). His‐PilMK26A and PilN‐Strep were coproduced in Escherichia coli and copurified from solubilized membranes via Strep‐Tactin affinity chromatography. One microgram of purified protein was separated in 14% SDS/PAGE. The dotted line indicates an excised lane from the same gel (D). His6‐PilMK26AN‐Strep pulled down PilF when co‐incubated on a Strep‐Tactin column as described in Fig. 2 (E). PilMK26AN stimulates PilF ATPase activity similar to wild‐type PilMN. PilF hexameric complexes were mixed with PilMN or PilMK26AN in a molar ratio of 1 : 20 for ATPase assays (F). Shown are means ± SD of at least three independent measurements.
Figure 6
Figure 6
The PilF GSPIIC domain is necessary for PilMN‐mediated stimulation but not for interaction. Co‐elution of PilMN with PilFK654A, PilFE718A and PilFΔGSPII variants. Dotted lines indicate excised lanes from identical gels (A). ATPase assay of PilFΔGSPII variants alone (light bars) and in presence of PilMN in a ratio of 1 : 20 (dark bars) as described above (B). Bars represent means ± SD of at least three independent measurements.
Figure 7
Figure 7
Model of the inner membrane platform interacting with the traffic ATPase PilF. PilN and PilO interact at the periplasmic face of the inner membrane (IM). The cytoplasmic N terminus of PilN interacts with the cytoplasmic PilM. PilM, when bound to PilN, stimulates activity of the soluble ATPase PilF. The energy provided by hydrolysis of ATP is then transmitted from PilF via the inner membrane platform comprising PilM, PilN, and PilO to further components of the DNA translocator/T4P machinery leading to DNA uptake and T4P assembly.

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