Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures - PubMed (original) (raw)

S Krause et al. Proc Natl Acad Sci U S A. 2000.

Abstract

RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop). Helicobacter pylori HP0525 also belongs to the PulE superfamily and is encoded by the pathogenicity island cag, involved in the inflammatory response of infected gastric epithelial cells in mammals. The native molecular masses of TrbB and HP0525 as determined by gel filtration and glycerol gradient centrifugation suggested a homohexameric structure in the presence of ATP and Mg(2+). In the absence of nucleotides and bivalent cations, TrbB behaved as a tetramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring-shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg(2+). However, the TrbB average image appeared to be more elliptical with strong twofold rotational symmetry whereas HP0525 complexes are regular hexagons. Six well defined triangle-shaped areas of high electron density were distinguishable in both cases. Covalent crosslinking of TrbB suggests that the hexameric ring is composed from a trimer of dimers, because only dimeric, tetrameric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.

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Figures

Figure 1

Figure 1

Alignment of VirB11 family transport NTPases. Amino acid sequences were aligned by using the

pileup

program of the GCG program package version 9.1.

boxshade

3.31 (

http://www.ch.embnet.org/software/BOX\_form.html

) was used with the following parameters: red background and white letters, 100% conservation; green background and white letters, at least 75% conservation of identical residue; and yellow background, at least 75% conservation of similar residues. The extensions of the four common motifs are marked by horizontal lines. Accession numbers: TraG (pKM101), I79275; TrwD(R388), X81123; VirB11 (pTi15955), X06826; VirB11 (pTiC58), P07169; VirB11 (Rhizobium etli), AF176227; VirB11 (Bartonella henselae), AF182718; PtlH (B. pertussis), F47301; VirB11 (Brucella suis), AF141604; RP292 (Rickettsia prowazekii), AJ235271; HP0525 (Helicobacter pylori cag), AE00148; TrbB (RP4), M93696; TrbB (R751), U67194; TrbB (pTiA6NC), P54907; TrbB (Rhizobium sp. NGR234), AE000068; Kbh (pCl1 Chlorobium limicula), U77780; and VirB11 (pXO1 Bacillus anthracis), AF065404.

Figure 2

Figure 2

Glycerol gradient centrifugation of TrbB and HP0525. Purified TrbB (A) [fraction IV, 150 μl, 435 μg; (20)] and purified HP0525 (B) [fraction IV, 150 μl, 435 μg; (20)] were laid on a 3.7-ml, 15–35% (wt/vol) linear glycerol gradient in buffer E or F. Centrifugation was at 270,000 × g for 15 h at 4°C. Before centrifugation, TrbB and HP0525 were incubated in buffer F for 10 min at 30°C. Fractions were tested for dATPase activity as published elsewhere (20, 30). Aliquots were electrophoresed in SDS/15% polyacrylamide gels. Gels were stained with Serva Blue R and scanned in the Personal Densitometer (Molecular Dynamics). The amount of protein was quantified by using the

imagequant

software. The stained gels of the glycerol gradient centrifugation in the absence of dATP and Mg2+ are shown in a (TrbB) and d (HP0525). Corresponding graphs are shown above the gels. Gels c (TrbB) and e (HP0525) are from glycerol gradient centrifugation in the presence of dATP and Mg2+. Corresponding graphs are shown below the gels. TrbB was also incubated with dADP and Mg2+, and glycerol gradient centrifugation was carried out in the presence of dADP and Mg2+ (b). In all graphs shown, open squares represent the ATPase activity of the fractions, filled squares, the amount of protein. Scales are given on the left and right hand sides of the graphs. Catalase (1: 240 kDa, _s_20,w = 11.3 S), aldolase (2: 185 kDa, _s_20,w = 7.8 S), and BSA (3: 68 kDa, _s_20,w = 4.4 S) were run in parallel as standards and marked by arrows.

Figure 3

Figure 3

Covalent crosslinking of TrbB and HP0525 subunits. TrbB protein (5 μM) and 15 μM HP0525, respectively, were incubated with various concentrations of glutaraldehyde in buffer G at 25°C for 90 min. Reactions were stopped with 3.5 M urea (final concentration). Denatured samples were electrophoresed in an SDS/10% polyacrylamide gel. Gels were stained with Serva Blue R. (A) Crosslinking of TrbB. Lanes: a, molecular mass markers; b, no glutaraldehyde; c, 100 μM; and d, 300 μM glutaraldehyde, respectively. (B) Crosslinking of HP0525. Lanes: a, molecular mass markers; b, no glutaraldehyde; c, 650 μM; and d, 1,300 μM glutaraldehyde, respectively.

Figure 4

Figure 4

Typical electron micrographs of negatively stained TrbB (a) and HP0525 (b) samples in the presence of Mg2+ and ATP. The arrrowheads point to some characteristic particles selected from these fields. The bar represents 50 nm. Two galleries with the type of images that were used in the two-dimensional analysis are shown in c (TrbB) and d (HP0525). The global average images are shown in e (TrbB) and f (HP0525) and have been low-pass filtered to their final resolution (14 Å in both cases). The size of the frames of each image in the galleries and for the final average images is 20.0 × 20.0 nm.

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