Comparison of the VacA p55 Structure with Structures of Other Autotransporter Passenger Domains.

Crystal structures have been determined for two other type Va autotransporter passenger domains: pertactin (an adhesin from Bordetella pertussis) and Hbp (a hemoglobin protease from Escherichia coli). Pertactin (41), Hbp (42), and VacA p55 do not share sequence similarities, but all contain a β-helix fold. The β-helix fold is composed of multiple ≈25-aa quasirepeats, each of which forms a single coil of the helix. Conservation of the β-helix fold among autotransporter passenger domains suggests that this structural feature is required for efficient secretion across the outer membrane and folding (43). A C-terminal β-helix cap may be important as a nucleus and/or chaperone for secretion and folding (44). The end of the VacA p55 β-helix is capped by a β-hairpin, similar to that observed in the structure of pertactin (41). There is also a short α-helix in this region of VacA that appears to be unique among β-helix structures (Fig. 1c).

A notable difference between VacA p55 and the two other autotransporter passenger domain structures is the presence of a disulfide in the p55 C-terminal subdomain (Fig. 1c). The low cysteine content observed in autotransporter sequences is consistent with a model in which passenger domains translocate across the outer membrane in an unfolded conformation. Nevertheless, a number of autotransporter sequences have a single, closely spaced pair of cysteines near the C terminus of their passenger domains (45). In VacA, these cysteines are either 11 or 13 aa apart. Mutation of either of these cysteines to serine results in a decrease in toxin secretion but has no effect on the vacuolating activity of the toxin (ref. 45 and M.S.M., unpublished results). Here, we show experimentally that these cysteines form a disulfide and that they are positioned to buttress both the β-helix cap and a conserved C-terminal pocket (discussed below). Another feature of p55 that differs from other autotransporter passenger domains is the presence of multiple kinks that disrupt what would otherwise be continuous β-sheets. We have divided the VacA p55 β-helix into five subdomains to reflect these disruptions (Fig. 2a) and note that the divisions correlate with predicted sites of homologous recombination (as discussed further below).

Open in a separate window

Fig. 2.

The VacA p55 structure reveals marked disruptions in β-sheet contacts. (a) Breaks in the β-sheet contacts (depicted with double-headed arrows) result in subdomains (SD) within the β-helix: SD-1 (red, 355–454), SD-2 (orange, 455–492), SD-3 (yellow, 493–615), SD-4 (purple, 616–647), and SD-5 (blue, 648–735). The C-terminal domain and disulfide bond are colored green and red, respectively, and the site of the m2 23-aa insertion is indicated by an asterisk. (b) This schematic shows prototype m1 and m2 VacA proteins (strains 60190 and Tx30a, respectively) and two naturally occurring m1m2 chimeras. The breaks in β-sheet contacts (a) correlate with sites of homologous recombination (see also SI Fig. 7). In this schematic the midregion that distinguishes type m1 and type m2 sequences is colored orange/yellow/purple/blue (m1) or black (m2).

Sequence Variation Among VacA Proteins.

In an effort to understand how genetic differences between m1 and m2 vacA alleles relate to structural and functional differences in the proteins they encode, we have examined VacA sequence polymorphisms in the context of the p55 structure. We identified sequences for 62 m1 and 27 m2 VacA proteins in GenBank that were complete in the p55 region. In addition, we identified three m1/m2 chimeric VacA proteins for which sites of recombination between m1 and m2 sequences were easily recognizable. We aligned these sequences in various combinations using the program ClustalW (46) and mapped the sequence similarity scores to the three-dimensional m1 p55 structure using ESPript (47). The majority of residues pointing into the interior of the β-helix are either strictly or highly conserved, consistent with the idea that mutation of buried residues would result in a detrimental loss of structure and, therefore, function. The surface-exposed residues are also fairly conserved when exclusively m1 or exclusively m2 sequences are analyzed (SI Fig. 6). However, when m1 and m2 sequences are analyzed together, the surface-exposed residues are highly variable (Fig. 3). There are only two regions of the surface with notable sequence conservation. One is located at the N terminus (Fig. 3a) and will be discussed in the next section with respect to protein oligomerization. The second conserved surface is located at the C terminus of the β-helix in a cavity formed by two loops (residues 668–678 and 730–734) and the disulfide-linked C-terminal domain (Fig. 3b). The strict sequence conservation, the fact that many binding sites are located in clefts or cavities, and the fact that this is the only cavity observed in the VacA p55 structure suggest that this area may represent a receptor-binding site that is shared by m1 and m2 forms of the toxin.

Open in a separate window

Fig. 3.

The VacA p55 structure has two patches of conserved residues. (a) The 62 m1, 27 m2, and 3 m1/m2 chimeric VacA sequences were aligned and scored with a Risler matrix according to the extent of sequence variation. Scores were displayed on the p55 structure surface with a color ramp (red, orange, yellow, green, light blue, dark blue) in which strictly conserved residues are colored red, and the most variable residues are colored dark blue. One conserved region is at the N terminus of the protein (boxed) and correlates to the β3, β6, and β9 strands of the β-helix. We propose that this surface is important for VacA oligomerization. (b) Rotation of the molecule in view a by ≈180° around the long axis of the protein reveals the second conserved region of the VacA p55 structure. This surface is located in a pocket at the C terminus of the β-helix (circled) and may represent a common receptor-binding site for all m1 and m2 VacA proteins. The pocket is formed by two long β-helix loops (red) and the disulfide bond (also in red) of the C-terminal domain. Although the asterisked region is also highly conserved, this correlates to the N terminus of the β-helix that we predict to be buried when p33 is present.

Because multiple surface-exposed regions are highly divergent, it is difficult to identify a single region in the p55 structure that can explain the different binding properties of m1 and m2 VacA proteins (Fig. 3). The biggest difference between m1 and m2 sequences is the presence of a 23-aa insertion in m2 sequences (Fig. 2a, asterisk; and SI Fig. 7). The insertion is notable in that its sequence is an imperfect repeat of the sequence located immediately upstream, and the length approaches the average length of a single β-helix coil (≈25 aa) (SI Fig. 7). The 23-aa m2 insertion could therefore result in an additional β-helix coil similar in structure to the coil that precedes it. Based on an analysis of engineered m1/m2 chimeras, a region important for m1-specific binding to HeLa cells has been mapped to residues 460–496 in the m1 sequence (37). The m2 23-aa insertion is located within this region, but an engineered chimera in which these 23 m2 amino acids were inserted into an m1 sequence did not result in a loss of HeLa cell toxicity (37). Excluding the m2 insertion, there are only 10 amino acids in the 460–496 m1 sequence that consistently differ between the m1 and m2 forms (SI Fig. 7). The position of these residues in the context of the 23-aa m2 insertion is impossible to predict, and, therefore, an understanding of structural features that give rise to VacA cell-type specificity may need to await analysis of an m2 p55 structure.

Analysis of the VacA sequence alignments in the context of the m1 p55 structure reveals that disruptions of β-sheet contacts within the β-helix correlate with sites of homologous recombination events. For example, VacA residue D455 (strain 60190) represents the beginning of the midregion that defines m1 and m2 strains (Fig. 2b and SI Fig. 7) and follows a kink in the structure that disrupts β-sheet contacts between β8 and β11 (Fig. 2a and SI Fig. 7). This structural disruption and the associated sequence transition are illustrated with a change from red to orange (Fig. 2 and SI Fig. 7). Other examples of a correlation between VacA subdomains and recombination events are evident based on analysis of rare naturally occurring m1/m2 chimeras, which have m1 character in the N-terminal portion of the VacA midregion but m2 character in the C-terminal portion. The transition point from m1 to m2 in the chimeric VacA from strain 249a3 is at D493, whereas the transition point for the ch2 and v225 strains of VacA is at N616 (Fig. 2b and SI Fig. 7). As illustrated in Fig. 2a, these sequence transitions correlate with disruptions in β-sheet contacts between β14 and β17 and between β29 and β32, respectively. The final subdomain begins at residue G648. Although we did not identify a sequence with evidence of recombination at this site, the subdomain (blue) has longer loops than other subdomains and does not make β-sheet contacts with β33 from the previous subdomain (Fig. 2a).

In summary, the polymorphisms that differentiate m1 and m2 forms correspond almost entirely to surface-exposed residues. Diversification in these surface-exposed regions of VacA may have been driven by immune selective pressures. Recombination occurs commonly in H. pylori (32, 33), but m1/m2 chimeras are only rarely identified (35). This suggests that H. pylori strains with intact m1 or m2 VacA sequences have favorable functional properties and, thus, have a selective advantage compared with strains containing chimeric m1/m2 sequences (36–38). The rare naturally occurring m1/m2 chimeras shown in Fig. 2b and SI Fig. 7 probably arose as the result of recombination events that maintained favorable structural and functional properties in the encoded β-helix structures.

Crystallization and Preparation of Heavy-Atom Derivatives.

The native and seleno-methionine p55 were concentrated in a 100 mM NaCl, 50 mM potassium phosphate buffer and crystallized at 21°C by the hanging-drop vapor-diffusion method in which protein and precipitant were mixed in a 1:1 ratio. Crystals grew from starting protein concentrations of 5.5 mg/ml, and reservoirs containing 18–23% PEG1500. Heavy-atom derivatives were prepared by soaking crystals in 50 mM potassium tetrabromoplatinate (IV) (K₂PtBr₆) for 3 d, 5 mM 1,4-diacetoxymercuri-2,3-dimethoxybutane for 24 h, or 5 mM gold chloride (HAuCl₄) for 24 h. Crystals were mounted on cryo loops, sequentially soaked in cryoprotectant solutions containing 25% PEG1500, and 5%, 10%, and 15% glycerol, and flash-cooled in liquid nitrogen.

We thank Catherine El-Bez and Jacques Dubochet for providing the cryo-EM map. X-ray data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory (Argonne, IL). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. This work was also supported by National Institutes of Health Grant R01 AI39657 (to T.L.C.), the Medical Research Service of the Department of Veterans Affairs (T.L.C.), the Sartain-Lanier Family Foundation and an American Cancer Society Institutional Research Grant IRG-58-009-48 (to D.B.L.), and the T32 GM 08320 Molecular Biophysics Training Program (K.A.G.).