Crystal Structure of the Hexameric Traffic ATPase of the Helicobacter pylori Type IV Secretion System

Molecular Cell, Vol. 6, 1461–1472, December, 2000, Copyright 2000 by Cell Press

Crystal Structure of the Hexameric Traffic ATPase of the Helicobacter pylori Type IV Secretion System Hye-Jeong Yeo,*§ Savvas N. Savvides,*§ Andrew B. Herr,*k Erich Lanka,† and Gabriel Waksman*‡ * Department of Biochemistry and Molecular Biophysics Washington University School of Medicine St. Louis, Missouri 63110 † Max Planck Institut fu ¨ r Molekulare Genetik Ihnestrasse 73, Dahlem D-14195 Berlin Germany

Summary The type IV secretion system of Helicobacter pylori consists of 10–15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release. Introduction Pathogenicity in gram-negative bacteria is critically dependent upon secretion machineries that mediate the transport and injection of toxic molecules into target cells (Finlay and Falkow, 1997; Thanassi and Hultgren, 2000). These secretion systems are classified into four types (I to IV) and share a common requirement for proteins that utilize ATP as an energy source to drive transport of macromolecules (Salmond, 1994). One such class of ATPase is that of the PulE-like proteins that comprise ATPases involved in both type II and type IV secretion (Possot and Pugsley, 1994; Russel, 1998; Christie and Vogel, 2000). Pathogenic bacteria that utilize type II secretion systems include human pathogens, such as enteropathogenic Escherichia coli (EPEC) causing infant diarrhea; Vibrio cholerae causing cholera; Pseudomona aeruginosa, a leading cause of mortality in compromised patients with cystic fibrosis; or Neisseria gonorrhoea, which causes gonorrhea; as well as plant pathogens, such as Erwinia chrysanthemi or Xanthomonas campestris (reviewed in Finlay and Falkow, 1997). ‡ To whom correspondence should be addressed (e-mail: waksman@

biochem.wustl.edu). § These authors contributed equally to this work. kPresent address: Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125.

Type IV secretion systems are used, for example, by Helicobacter pylori, which causes peptic ulcer diseases; Legionella pneumophila, which causes pneumonia; or Bordetella pertussis, which causes whooping cough (Vogel et al., 1998; Burns, 1999; Covacci et al., 1999; Segal et al., 1999a). Type IV secretion systems are used not only to transport molecules toxic to host cells, but also are used to transport DNA or protein–DNA complexes (Winans et al., 1996). One such process is bacterial conjugation whereby two mating bacteria exchange genetic material (Lanka and Wilkins, 1995; Pansegrau and Lanka, 1996). By facilitating conjugative transfer, type IV secretion machineries play crucial roles in the spread of antibiotic resistance genes among bacteria. Another process utilizing type IV secretion systems for nucleoprotein transport is the transformation of plants by Agrobacterium tumefaciens (de la Cruz and Lanka, 1998; Kado, 1998; Rossi et al., 1998). In that process, a fragment of the Ti (tumor-inducing) plasmid, the T-DNA, is transferred into the plant cell (de la Cruz and Lanka, 1998; Rossi et al., 1998). This transfer system has been exploited in plant biotechnology to introduce new genes conferring herbicide resistance or resistance to pathogens of industrial crops (D’Halluin and Botterman, 1998; Chua and Sundaresan, 2000). Type IV secretion systems require a subclass of PulEtype ATPases, which are known under the generic name of VirB11 ATPases (Motallebi-Veshareh et al., 1992). VirB11 ATPases are essential for T-DNA transfer in A. tumefaciens, conjugative transfer of DNA in most systems studied to date, and protein transfer in H. pylori, L. pneumophila, and B. pertussis (Haase et al., 1995; Stephens et al., 1995; Vogel et al., 1998). Despite the fact that VirB11 ATPases are known to be essential for type IV secretion and pathogenicity, little is known about the function that these proteins fulfill in these machineries (Christie and Vogel, 2000). Their role may vary from one organism to another depending on the type of transport they help mediating. For example, in the conjugative system encoded by the RP4 plasmid, the VirB11 ATPase, TrbB, is known to be involved in pilus biogenesis (Haase et al., 1995). Indeed, many type IV secretion systems, notably those involved in conjugative transfer and T-DNA transfer, function in conjunction with a fibrous cell surface organelle called “pilus,” which is thought to be important for adhesion between bacteria during conjugative transfer or between bacteria and host eukaryotic cells during infection (Eisenbrandt et al., 1999; Soto and Hultgren, 1999; Lai and Kado, 2000). In H. pylori, however, there is no known pilus associated with the type IV secretion system. As a result, the VirB11 ATPase of H. pylori may be directly involved in transport of proteins through the inner membrane. In either case, the potential roles of VirB11 proteins are consistent with evidence that these proteins are at least in part associated with the inner membrane (IM) and localized to the cytoplasmic side of the IM (Thorstenson et al., 1993; Rashkova et al., 1997; Grahn et al., 2000). Recently, some of the biochemical properties of three

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Figure 1. Structure of the HP0525–ADP Complex (A) Representative region (the ␤6 strand) of the experimental electron density at 3 A˚ resolution. The electron density results from a map calculated using SAD phases after NCS averaging and solvent flipping and is contoured at a 2␴ level. The final refined model is shown in stick representation color-coded in yellow for carbon, blue for nitrogen, and red for oxygen. (B) Stereo ribbon diagram of the structure of HP0525 bound to ADP and a 9-mer of PEG (Carson, 1997). ␣ helices, 310 helices, strands, and loops are indicated in blue, dark blue, green, and amber, respectively. The ADP is in magenta, and the PEG molecule is in gray. Secondary structural elements (except for the 310 helices) are labeled from ␤1 to ␤13, while helices are labeled from ␣A to ␣I. N and C indicate the N and C termini of the protein, respectively.

VirB11 ATPases have been reported, those of the RP4 and R388 plasmid conjugative systems, TrbB and TrwD, respectively, and that of the type IV secretion system from H. pylori, HP0525 (Krause et al., 2000a, 2000b). The nucleotide requirements vary from one protein to another. However, ATPase activity is in all cases stimulated by lipid binding, consistent with the localization of VirB11 ATPases to the membrane. More importantly, these proteins form hexameric ring structures (ⵑ12 nm in diameter) of identical subunits reminiscent of structures of ATP-dependent molecular motors, such as helicases or F1-ATPases (Krause et al., 2000a; Patel and Picha, 2000). These structures suggest that VirB11 proteins may actively engage in translocation of substrates. In this report, we describe the crystal structure of the VirB11 ATPase, HP0525, from H. pylori bound to ADP. H. pylori is the causative agent of gastric ulcers, a widespread disease in both the developed and developing worlds. H. pylori is probably the most common chronic bacterial infection in humans, present in almost half of the world population (Covacci et al., 1999). The presence of the bacterium in the gastric mucosa is associated with chronic active gastritis and is implicated in more severe gastric diseases, including chronic atrophic and mucosa-associated lymphoid tissue (MALT) lymphomas (Wotherspoon, 1998; Graham, 2000). One substrate for secretion is a 145 kDa protein, CagA (Segal et al., 1999b; Asahi et al., 2000; Backert et al., 2000; Odenbreit et al., 2000; Stein et al., 2000). CagA is injected into gastric or duodenal epithelial cells by the type IV secretion system encoded by the Cag pathogenicity island. CagA, once injected, becomes tyrosine-phosphorylated and is thought to interfere with normal signaling in host cells. The crystal structure of HP0525 sheds light on the func-

tion of HP0525 and provides the basis for designing antiulcer drugs. HP0525 resembles a six-clawed grapple mounted onto a hexameric ring structure. The state captured in the crystal structure is a closed form where the six grapple elements are clawed together to form a chamber that is open on the hexameric ring side and closed on the grapple side. The chamber is large enough to accommodate a large protein, such as CagA. A model is proposed as to how proteins could be transported through the ring structure by concerted opening of the six-clawed grapple upon release of nucleotide. Result and Discussion Structure of the HP0525 Monomer The structure of the HP0525 monomer bound to ADP is shown in Figure 1. The structure contains two domains that are formed by two contiguous parts of the amino acid sequence (Figure 2). The N-terminal part from residue 6 (the first residue for which interpretable electron density was observed) to residue 136 forms the N-terminal domain, while residues 137 to 328 (the last residue for which interpretable electron density was observed) form the C-terminal domain. Topologically, the fold adopted by the C-terminal domain is that of the RecA protein (Story et al., 1992). In contrast, submission of the coordinates of the N-terminal domain to the DALI server did not return a known fold, suggesting that the fold adopted by the N-terminal domain is novel (Holm and Sander, 1998). Each domain is composed of an extended central ␤ sheet containing six ␤ strands in the N-terminal domain and seven ␤ strands in the C-terminal domain. While the central ␤ sheet of the C-terminal domain is flanked on both sides by ␣ helices (three on one

Structure of the VirB11 ATPase of H. pylori 1463

Figure 2. Sequence Alignment of the VirB11 ATPases and Location of Secondary Structures in H. pylori HP0525 The following sequences were used in the alignment: HP0525 from H. pylori, LvhB11 from L. pneumophila, VirB11 from A. tumefaciens, TrbB from plasmid RP4, and TraG from plasmid pKM101. Strictly conserved residues are shown in green boxes, while residues that are conservatively substituted are shown in pink boxes. Amino acid numbering at the top refers to HP0525. Numbering at the end of the lines refers to the number of the last amino acid shown at the end of the line in the corresponding sequence. Secondary structural elements of HP0525 are shown at the bottom with helices and strands indicated as cylinders and arrows, respectively. 310 helices are not shown for clarity. Residues involved in subunit–subunit interactions are shown in blue boxes. Residues involved in ADP binding and proposed to be involved in ATP binding and hydrolysis are boxed in red.

side [␣C, ␣D, and ␣E] and four on the other [␣F, ␣G, ␣H, and ␣I]), that of the N-terminal domain is packed against two ␣ helices on only one side (␣A and ␣B). One of these two ␣ helices (helix ␣A) extends far out away from the core of the protein. The side of the six-stranded central ␤ sheet opposite the two flanking ␣ helices in the N-terminal domain forms a polar surface that is extensively used as a subunit-subunit interface in the hexamer. The two domains are linked by a short loop between residues 134 and 141. This linker region emanates from the ␤6 strand in the N-terminal domain, which is in an antiparallel arrangement with the two strands preceding it (strands ␤5 and ␤4; Figure 1B). On the other side, the ␤6 strand runs antiparallel to the ␤1 strand, which itself forms an antiparallel ␤ sheet with the two strands succeeding it (strand ␤2 and ␤3; Figure 1B). A long helix, ␣B, spans the length of the entire six-stranded ␤ sheet in the N-terminal domain such that its N terminus connects to the edge strand ␤3 on one side, and its C terminus connects to the other edge-strand ␤4 on the other side. The N-terminal helix, from residue 6 to residue 40, is bent at two positions, at residue 12 and at residue 35. The ␣A helix is followed by a short loop connecting to the ␤1 strand. The first secondary structures in the C-terminal domain are helices ␣C and ␣D. Helix ␣D is the first secondary structural element that aligns with the RecA protein, as do most of the subsequent secondary structures from

strand ␤7 to strand ␤13 (Story et al., 1992). Only ␣ helices ␣H and ␣I have no equivalent in the RecA fold. These two helices protrude from the core structure of the domain, and together with the same regions of the other subunits form the narrow end of the hexameric structure (see below for details). The region between strands ␤8 and ␤9 also differs significantly from the RecA structure: while these two strands are connected by an extended loop in HP0525, the equivalent region in RecA contains three ␣ helices (Story et al., 1992). Structure of the HP0525 Hexamer The HP0525 hexamer can be readily assembled using one of the two 3-fold crystallographic symmetry axes intrinsic to the hexagonal space group into which the protein–ADP complex crystallizes. A ribbon diagram representation of the resulting hexameric structure is shown in Figure 3 while a surface diagram onto which the surface electrostatic potential of the hexameric assembly has been mapped is shown in Figure 4. The six HP0525 subunits assemble in an intertwined propeller shape whereby residues in both domains of individual subunits participate in the subunit–subunit interface. The entire interface amounts to 2260 A˚2 of buried surface area. The overall shape of the hexamer is that of a sixclawed grapple mounted on a hexameric ring. Indeed, when each domain is color-coded differently as is shown

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Figure 3. Ribbon Diagrams of the HP0525 Hexamer In (A), (B), and (D), each HP0525 subunit is color-coded differently. Each is represented in ribbon diagram as in Figure 1B. Note that the subunit in yellow in (D) is in the same orientation as in Figure 1B. ADP is shown in magenta, while PEG is shown in gray. In (C), each subunit is in the same color, with the N-terminal domain colored in gold and the C-terminal domain colored in magenta. In (C), the ADP is shown in dark blue and the PEG is cyan. (A) Stereo ribbon diagram of HP0525 viewed down the large hole formed by the N-terminal domains (Carson, 1997). (B) Stereo ribbon diagram of HP0525 viewed from the side. This view is obtained by rotating the HP0525 shown in (A) 90⬚ clockwise along the vertical axis of (A). (C) Ribbon diagram of HP0525 in the same orientation as (B). The color coding of the subunit illustrates the double-ring structure of HP0525. The labeling above the panel indicates the putative location of the surfaces spanning the inner membrane (IM) or exposed to the cytoplasm (Cyt). (D) Stereo ribbon diagram of HP0525 viewed down the small hole at the base of the grapple formed by the tips of the C-terminal domains.

Structure of the VirB11 ATPase of H. pylori 1465

Figure 4. Surface of the HP0525–ADP Complex The surfaces were contoured and displayed using the program GRASP (Nicholls et al., 1991). Color coding is according to charge, blue for the most positive regions and red for most negative regions with linear interpolation in between. (A) Surface of the HP0525–ADP complex viewed down the large hole. This view corresponds to that of Figure 3A. The inside and outside dimensions of the HP0525 chamber are indicated in yellow. (B) Surface of the HP0525–ADP complex viewed from the side. Top, the entire HP0525 hexamer. This view corresponds to that shown in Figures 3B and 3C. The putative location of the membrane-spanning and cytoplasm-exposed regions of the HP0525 hexamer are indicated. Also three 9-mer PEG molecules and two ADP molecules are clearly visible and shown as ball-and-stick representation (white for carbon, red for oxygen, yellow for phosphorus, and blue for nitrogen) and labeled accordingly as PEG and ADP. Bottom, cut-away of the HP0525 hexamer. The orientation is the same as in the top. Four subunits are shown, which allow to visualize the surface potential inside the HP0525 chamber. The inside and outside dimensions of the HP0525 chamber are indicated in yellow. (C) Surface of the HP0525–ADP complex viewed down the small hole of the chamber.

in Figure 3C, it is apparent that the N-terminal domains and the C-terminal domains form two separate ring structures. The N-terminal domains form a hexameric ring that is cylindrical inside (Figures 3A and 3B). In contrast, the C-terminal domains form a hexameric ring that is conical inside and where each of the C-terminal domains looks like a grapple claw (Figures 3A and 3D; Figures 4A and 4C). This comparison facilitates the understanding of our proposal that HP0525 may act as a protein translocator. The HP0525 hexamer has an external diameter of 100 A˚ and an internal diameter of 50 A˚ (Figure 4A). The outside cross section (outside, from top to bottom) of the hexamer is 50 A˚, while its inside cross section (inside, from top to bottom) is 30 A˚ (Figure 4B). The small hole formed by the closed grapple claws is 10 A˚ in diameter. Hence, a chamber of about 60,000 A˚3 is formed, open on one side (the side of the ring formed by the N-terminal domains) and closed on the other side (the side of the

ring formed by the C-terminal domains). Such a chamber is large enough to accommodate a globular protein or protein domain of about 50 kDa. A 9-mer of polyethylene glycol (PEG) was found bound to each of the N-terminal domains at the periphery of the hexameric ring (Figure 1B). The presence of PEG bound to these surfaces suggests that the N-terminal domain ring may be embedded into the membrane. VirB11 ATPases are known to be associated to the membrane. We propose here that membrane association is mediated by contacts with the mostly hydrophobic ring formed by the N-terminal domains. While the outside surfaces of the cylindrical hexameric N-terminal domain ring and part of the C-terminal domain ring juxtaposed to it are relatively hydrophobic, the tip of the C-terminal domain ring facing the outside (the cytoplasmic face) is characterized by a strong negative electrostatic potential (Figure 4C). Hence in the closed configuration of the grapple reported here, nega-

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Figure 5. The Subunit–Subunit Interface of the HP0525 Hexamer In (A) through (D), the two subunits are colorcoded red and yellow, respectively, and represented as ribbons as in Figure 3A. Secondary structural elements are labeled according to the nomenclature defined in Figure 1B. (A) Overview of the subunit–subunit interface (Carson, 1997). Residues involved in interactions between subunits are shown in balland-stick representation with residues responsible for contacts between N-terminal domains indicated in green, residues involved in interactions between the N-terminal domain of one subunit and the C-terminal domain of the other subunit indicated in cyan, and the residues involved in contacts between C-terminal domains indicated in magenta. (B) Stereo diagram of the interface between N-terminal domains (the region indicated in green in [A]). Residues are in balland-stick representation color-coded gray for carbons, red for oxygen, blue for nitrogen, and green for sulfur. Residues are labeled, as are the few of the secondary structural elements shown. (C) Stereo diagram of the interface between N-terminal and C-terminal domains of adjacent subunits (the region indicated in cyan in [A]). Residues are shown and labeled as in (B). ADP is shown in ball-and-stick representation color coded in magenta. The black circle locates a large cluster of arginine residues believed to destabilize the interface in the absence of nucleotide. (D) Stereo diagram of the interface between C-terminal domains (the region indicated in magenta in [A]). Residues are shown and labeled as in (B).

tive charges would face the cytoplasm, capping the HP0525 hexamer and acting as either repelling or attractive forces for a yet unidentified binding partner or substrate. If, as discussed below, one hypothesizes that opening of the grapple is required for uptake of proteins, then such surfaces would act either by binding to a partner facilitating opening (possibly CagA itself, which has a pI of 9.2 and therefore would be positively charged in the pH environment of the membrane) or by repelling a factor facilitating closure (possibly the head groups

of the lipid bilayer when the grapple is in its open conformation). The inside of the HP0525 ring is characterized chemically by a mixed composition of predominantly hydrophobic patches and a few negatively and positively charged surfaces (Figures 4A and 4B). Hence, if the grapple were to open, an only moderately positively charged surface would now be facing the cytoplasm. This surface would likely be the interacting surface for any protein presented to the resulting open pore.

Structure of the VirB11 ATPase of H. pylori 1467

Figure 6. The Nucleotide Binding Site (A) Interaction diagram between the ADP (in magenta) and the protein (in black). Residues involved are labeled. Interactions are indicated by dashed lines with distances between atoms involved in the interactions indicated in angstroms. (B) Stereo ribbon diagram of the ADP–protein interactions (Carson, 1997). The protein is represented as a red ribbon with secondary structures defined as in Figure 1B. The ADP is in magenta. Residues involved in interactions are in ball-and-stick representation color coded as in Figure 5B. Two residues, Glu248 and Glu-209, are shown in pink. These residues do not interact with ADP; however, their positions within the nucleotide binding site suggest that they act as catalysts for ATP hydrolysis.

The Subunit–Subunit Interface To form the hexamer, each HP0525 subunit interacts with two flanking subunits. Each subunit–subunit interface is extensive, burying a surface of 2260 A˚2. The interface between two subunits is shown in Figure 5A: it comprises contacts between residues in the N-terminal domains (in green in Figure 5A), between residues in the C-terminal domain of one subunit and the N-terminal domain of the adjacent subunit (in cyan in Figure 5A), and between residues in the C-terminal domains (in magenta in Figure 5A). These three interfaces are described separately below and are shown in the corresponding Figures 5B to 5D. As will be apparent in the description below, a striking feature of the interface used to assemble the HP0525 hexamer is its polar and charged nature. Contacts between N-terminal domains for the most part involve residues in the ␣A helix of one subunit with residues in the ␣B helix and in the ␤5-␤6 loop of the

other subunit (Figure 5B). Two salt bridges are formed: one between Arg-18 (in ␣A) and Asp-125 and Glu-126 (in the ␤5-␤6 loop), and the other between Glu-9 (in ␣A) and Lys-74 (in ␣B). Hydrophobic contacts are also observed between Phe-13 and Leu-14 on one side (␣A) and Leu-78 on the other side (␣B). Overall, this interface is small (463 A˚2). The bulk (1372 A˚2) of the interactions in the subunit– subunit interface is made between the N-terminal domain of one subunit and the C-terminal domain of the adjacent subunit (Figure 5C). A remarkable feature of the interface in this region is that most of the solventaccessible surface in the six-stranded ␤ sheet of the N-terminal domain is used. The N-terminal domain contributes residues from all strands except for the edge strand ␤4 (Figure 5C). These residues are involved in contacts with a region of the C-terminal domain that comprises the edge strand ␤9 of the central seven-

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stranded ␤ sheet, helix ␣F, and the loops between helix ␣F and strand ␤10 and between helix ␣E and strand ␤8. Here again, polar and ionic interactions are numerous. Two tryptophans, Trp-57 (in ␤2) and Trp-65 (in ␤3), are involved in extensive contacts with residues in the ␤8␤9 loop and in the ␣E-␤8 loop. Also involved in the interface is a continuous stretch of residues from Tyr218 to Phe-222 (i.e., residues in the entire ␤9 strand of the C-terminal domain are implicated in subunit–subunit assembly). Another remarkable feature of the N-terminal/C-terminal domains interface region is the involvement of a cluster of basic and acidic residues located near the ATP binding site (indicated in a circle in Figure 5C; also note the location of the ADP molecule in magenta in Figure 5C). Residues in contact are on one side Arg238 and Arg-240, and in the other side Glu-47, Arg-113, and Arg-133. While Glu-47 makes ionic interactions with Arg-238, the side chains of Arg-113, Arg-240, and Arg133 would clash electrostatically if it were not for the presence of the bound ADP molecule. Indeed, Arg-133 interacts directly with the ␣- and ␤-phosphate groups of the ADP (Figure 6), and the ␤-phosphate group is 5.5 A˚ away from the guanido group moiety of Arg-240. A modeled ␥-phosphate (not shown) would interact directly with Arg-240. Only the charge emanating from Arg-113 would not apparently be neutralized: its guanido group is 7.3 A˚ away from the ␤-phosphate of ADP. However, this side chain is solvent exposed and could readily undergo a local conformational change that would bring it within contact with the modeled ␥-phosphate of ATP. Thus, ADP or ATP would appear to be crucial to neutralize the like-charge clashes in this region. We propose that in the absence of the nucleotide, such clashes would have a destabilizing effect on the whole interface leading to the release of the C-terminal domain from the interface and its swiveling to an open conformation. Finally, contacts are made between the C-terminal domains of two adjacent subunits (Figure 5D). The interface in this region buries 425 A˚2 of surface area. Interactions are between the claws of the grapple and possibly help maintaining the grapple in the closed conformation seen in the crystal. More specifically, interactions involve primarily residues in the C-terminal end of the ␣G helix of one subunit with the residues of the ␣H helix of the adjacent subunit. Here also, the contacts made are primarily polar. The Nucleotide Binding Site The nucleotide binding site is described in Figure 6. The ADP is located between the N-terminal and C-terminal domains, and residues in both domains contribute to the binding site. It is important to note that, without ADP bound, contacts between the two domains within one subunit would be very scarce. Indeed, there are only a few direct contacts between the two domains, and the contact area directly involving the two domains is a mere 90 A˚2. This implies that the nucleotide-mediated contacts between the two domains are the driving force maintaining the two domains together. One would therefore predict that, in the absence of nucleotide, the domain–domain interface within a subunit would not be sufficient to maintain the two domains in a specific relative orientation.

Figure 7. Schematic Model for the Opening and Closure of the HP0525 Chamber The N-terminal and C-terminal domains are represented in dark and light blue, respectively. The view is down the big hole as in Figure 3A. ATP molecules are indicated as yellow balls.

In the C-terminal domain, a predominant region involved in contact with the ADP is the P loop, which is located between strand ␤7 and helix ␣E. Interactions involve main chain amide groups of Lys-84, Gly-183, Ser-182, and Gly-181. The charged tip of Lys-184 coordinates the ␤-phosphate, while two threonines immediately C-terminal to the P loop (Thr-185 and Thr-186) make hydrogen bonding interactions with both the ␣- and ␤-phosphates. The C-terminal domain is also involved in contacts with the adenine moiety with the adenine ring lodged between Tyr-140, Phe-145, and the aliphatic moiety of Lys-320. Coordination of the ribose moiety of the ADP molecule is mostly carried out by residues in the N-terminal domain (Thr-46 and Asn-61). However, the N-terminal domain is also involved in coordinating the phosphate moiety of ADP through Arg-133 and Ser-136. In RecA-like proteins, ATP hydrolysis is catalyzed by acidic residues located within the ATP binding site. In RecA, two acidic residues have been implicated in catalysis: Glu-96 has been proposed to activate the attacking water during hydrolysis of ATP, while Asp-144 is thought to coordinate the magnesium ion (electron density for magnesium was not observed in the HP0525-ADP complex) (Story and Steitz, 1992). Both Glu-96 and Asp-144 have their equivalents in HP0525: those are Glu-209 and Glu-248, respectively. Thus, we propose that these two residues are involved in ATP hydrolysis in HP0525. As noted before, the ADP binding site is close to the subunit–subunit interface and is contiguous with a cluster of arginine residues contributed by both subunits. We postulate that this observation has functional significance since, in the absence of ADP or ATP, destabilization of the interface between the C-terminal domain of one subunit and the N-terminal domain of the adjacent subunit may occur leading to a possible release of the C-terminal domain from the interface. A Model for HP0525 Function VirB11 ATPases are known to be essential in several important processes, such as T-DNA transfer, conjugative transfer of many plasmids, and type IV secretion. VirB11 proteins are also homologous to PulE ATPases, which play crucial roles in type II secretion and type IV pilus biogenesis (not to be mistaken with type IV secretion, since the assembly machinery of type IV pili

Structure of the VirB11 ATPase of H. pylori 1469

Table 1. Data Collection, Phasing, and Refinement Data Collectiona Data Set

Resolution

Reflections (Total/Unique)

Completeness (%)

Rsym (%)b

I/␴(I)

SeMet-peak (all data) SeMet-peak (I/␴(I) ⬎ 0)

30–2.5 A˚ 30–2.5 A˚

163,385/29,176 137,723/27,352

94.7 (84.2) 88.8 (71.4)

10.8 (25.9) 10.2 (15.8)

10.8 (5.7) 10.7 (5.7)

Refinement Resolution 兩F兩 /␴(兩F兩) No. of reflections (working/test) Completeness (overall/last shell) Total No. of atoms Protein atoms ADP molecules PEG (n ⫽ 9) Water molecules R factor R-free factor Averaged B factors (A2) Main chain Side chain Wilson plot B (A2) Rms deviations NCS (A˚) Bonds (A˚) Angles (⬚) B values (A˚2) a b

30–2.5 A˚ (2.59–2.50 A˚) ⬎0 26,424 (1354) 89% (71%) 5,321 5,104 (646 residues) 2 1 300 0.223 0.289 36.0 36.3 38.0 0.09 0.010 1.50 1.6/2.5

Numbers in parentheses indicate values in the highest resolution shell (2.59–2.50 A˚). Rsym ⫽ 兺兩I ⫺ ⬍I⬎ 兩/兺I, where I equals observed intensity, and ⬍I⬎ equals average intensity for symmetry-related reflections.

is related to type II secretion systems). However, very little is known as to how they are implicated in these biological processes. Although, in the absence of biochemical data, any derivation of function from structure is speculative, one can infer from the structure of HP0525 bound to ADP a working model for the function of this protein in the type IV secretion system encoded by the Cag pathogenicity island of H. pylori. We propose here that HP0525 acts as a hexameric pore or portal, the closure and opening of which is regulated by concerted binding of ATP and release of ADP, respectively (Figure 7). We further propose that this ATP/ADP-controlled pore or portal is involved in translocating proteins, either the CagA protein itself or components of the type IV secretion machinery, through the inner membrane. Our proposal is based on the following observations: (1) there is little contact between the N-and C-terminal domains within a given subunit, and hence, each C-terminal domain is only stabilized by the extended polar interface that it forms with the N-terminal domain of the adjacent subunit; (2) the nucleotide is an important component of the subunit–subunit interface in that it neutralizes like charges, which, in its absence, would clash and possibly destabilize the whole interface in that region. A possible effect of these clashes would be to release the C-terminal domain from the interface, allowing it to swivel out to open up the chamber; (3) many properties of nucleotide binding in HP0525 are reminiscent of the way nucleotide binding and hydrolysis operate the opening and enlargement of the chamber of chaperones, such as GroEL/GroES (Sigler et al., 1998; Weber et al., 1998). Indeed, in HP0525, the nucleotide plays a critical role in the interface within and between

domains and its occupancy is similar in all subunits. Hence, subunits of HP0525 may operate simultaneously upon binding of ATP or release of ADP. The opening of the HP0525 chamber would create a large cylinder, which could accommodate passage of large proteins or protein domains. The CagA protein is certainly a potential candidate for transport. Another possibility, however, would be that HP0525 is utilized as an inner membrane usher to ferry the protein subunits required to build the parts of the type IV secretion machinery bridging the inner and outer membranes. Such a role is conceivable, since HP0525 is a homolog of the PulE class of ATPases, some of which, such as the PilF ATPase of N. gonorrhea, is required for macromolecular assembly of type IV pili (Freitag et al., 1995; Watson et al., 1996; Sauvonnet et al., 2000). In either case, these observations suggest that VirB11 and PulE ATPases serve as “traffic” ATPases, facilitating traffic of proteins through the inner membrane. VirB11 ATPases are also parts of machineries that transport nucleic acid (T-DNA transfer and conjugative transfer). Nucleic acids are in most cases transported as single-stranded (ss) DNAs coated with ssDNA binding proteins (Rees and Wilkins, 1989, 1990; Gelvin, 1998; Wilkins and Thomas, 2000). Hence, the transport of nucleic acid may not differ in fundamental ways from the transport of protein. It would however have to be a sequential unidirectional process, since ssDNA binding proteins form long clusters along the ssDNA, which resemble beads on strings (Gray, 1989; Griffith et al., 1984). Hence, continuous transport of a single string of several hundreds of ssDNA binding proteins bound to ssDNA may require an active system of translocation acting unidirectionally. In that context, the VirB11 ATPase ap-

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pears to be well suited to accomplish this task. Its chamber is large enough to accommodate one ssDNA binding protein/ssDNA complex, such as the one generated by E. coli SSB, for example (Raghunathan et al., 2000). Furthermore, one complex could be translocated and prevented from stepping back by the partial closure of the HP0525 chamber. In that case, sequential cycles of ATP binding and ADP release would serve to ratchet the ssDNA binding protein/ssDNA complexes through. There are to date no drugs targeting specifically type II or IV secretion machineries. Such drugs would have the advantage of applying to a narrow spectrum of organisms. The structure of HP0525 provides the basis for designing anti-ulcer drugs and could serve as a structural model for other ATPases of the PulE family to help design drugs targeting other important pathogens. Experimental Procedures Purification of Selenomethionyl HP0525 HP0525 (in plasmid pWP4760; Krause et al., 2000a) was expressed in the methionine auxotroph E. coli strain DL41 in the presence of selenomethionine using standard procedures. After sonication in a buffer containing 100 mM HEPES (pH 6.6), 200 mM NaCl, 2 mM EDTA, 2 ␮g/ml aprotinin, 2.5 ␮g/ml leupeptin, 1 mM PMSF, and 2 mM DTT, cell lysates were centrifuged at 15,000 rpm for 20 min. Proteins in the supernatant were precipitated by the addition of (NH4)2SO4 at 60% saturation on ice. After centrifugation, pellets were resuspended in 20 ml (per 1 liter culture) of buffer A containing 20 mM HEPES (pH 6.6), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and dialyzed against 2 liter of buffer A overnight. The dialyzed protein sample was applied to a 5 ml Hitrap Q column equilibrated in buffer B containing 20 mM HEPES (pH 6.6), 0.1 M NaCl, 1 mM DTT, 10% glycerol. The flowthrough contained selenomethionyl HP0525 as the only major protein and was dialyzed against buffer C (20 mM HEPES (pH 8.0), 0.1 M NaCl, 1 mM DTT, 10% glycerol) for at least 4 hr and loaded onto a second Hitrap Q column equilibrated in the same buffer. Selenomethionyl HP0525 eluted at 0.3 M NaCl. Pooled fractions were dialyzed against buffer D (20 mM MES [pH 7.0], 0.1 M NaCl, 1 mM DTT, 10% glycerol) and applied to a 2 ml hydroxyapatite column (BioRad CHT2 column) equilibrated in buffer D. Proteins were eluted with a 40 ml linear gradient from 0 to 0.35 M potassium phosphate (pH 7.0) in buffer D. Selenomethionyl HP0525 eluted at 0.17 M potassium phosphate. HP0525 was further purified by gel filtration (Sephacryl S-200 26/60 column, Pharmacia) equilibrated in buffer D. The protein is 99% pure at this stage. Crystallization of the HP0525–ADP Binary Complex The selenomethionyl HP0525–ADP complex was formed by mixing selenomethionyl HP0525 (0.5 mg/ml) with ADP-Mg to a final concentration of ADP-Mg of 5 mM. The complex was then concentrated to ⵑ30 mg/ml. Crystals of the selenomethionyl HP0525–ADP complex were grown at room temperature by vapor diffusion in hanging drops against a reservoir solution containing 100 mM Tris-HCl (pH 7.0–7.5), 19%–21% PEG1000, 0.2 M calcium acetate, and 15% glycerol (McPherson, 1990). Hexagonal-shaped plate crystals were formed within 24 hr and continued to grow for 1 week. Single crystals (0.3 ⫻ 0.3 ⫻ 0.07 mm) were flash-frozen to liquid nitrogen temperature. Crystals were in space group P6322 with cell dimensions a ⫽ b ⫽ 112.57 A˚, and c ⫽ 234.65 A˚ with two HP0525 subunits in the asymmetric unit. MAD data were collected to a resolution of 2.5 A˚ at three wavelengths using a single crystal (Beamline 19ID, Structural Biology Center, Advanced Photon Source). However, due to sharp decay during data collection, only the data collected at the first wavelength (the selenium absorption peak; Table 1) was usable, and the structure was solved using phasing based on single-wavelength anomalous diffraction (SAD). Structure Determination and Refinement The HP0525 monomer contains 7 methionines; hence, 14 seleniums were expected in the asymmetric unit. Anomalous difference Pat-

terson search (program CNS version 1.0; Bru¨nger et al., 1998) using the single wavelength peak data yielded 12 of the 14 possible sites. Inspection of the three-dimensional arrangement of these sites revealed two clusters of six selenium atoms related by a 2-fold noncrystallographic symmetry (NCS) axis parallel to the crystallographic c axis (hence this 2-fold axis was not observed in the self-rotation function). The 12 identified sites were then used in phasing using the SAD phasing method as implemented by the program CNS. Density modification (solvent flipping as implemented by the program SOLOMON; Abrahams and Leslie, 1996) resulted in a partly interpretable electron density map, into which 60% of the secondary structural elements could be placed as poly-Ala fragments (program O [Jones and Thirup, 1986; Jones et al., 1991]). From this partial model, a NCS mask was constructed that was used to refine the NCS operators previously derived from the relationship between selenium atoms using the program PHASES (Furey and Swaminathan, 1997). This mask and the refined NCS operators were then used in a protocol combining NCS averaging and solvent flipping (program CNS): the result was a greatly improved electron density map. Representative regions of this electron density map are shown in Figure 1. This map was used to place 90% of the secondary structural elements as poly-Ala. Several cycles of combining calculated phases from the minimized partial model with SAD experimental phases followed by NCS averaging and solvent flipping (program CNS) resulted in electron density maps that allowed building of a continuous poly-Ala chain for residues 7–328 and the identification of an ADP molecule. As phases continued to improve, side chains were gradually introduced. A thin tube of electron density located near the N-terminal domain became clearly apparent and was interpreted as a 9-mer polyethylene glycol molecule. The resulting atomic model was refined against 2.5 A˚ data using conjugate gradient minimization (program CNS). NCS restraints were applied to the main chain atoms of the two HP0525 molecules, and B factors were refined individually. Water molecules were added conservatively. After bulk solvent correction, the refinement converged to a final R factor of 22. 3% with an R-free factor of 28.9% (30–2.5 A˚ resolution range; |F|/␴(|F|) ⬎ 0.0) with good stereochemistry (Table 1) (Bru¨nger, 1992). The NCS restraints applied to the main chain atoms were such that the root-mean-square (rms) deviation between main chain atoms in the final model is 0.09 A˚. Average B factors were 36 A˚2 and 36.3 A˚2 for main chain and side chain atoms, respectively, and rms deviations for bonded atoms was 1.6 A˚2 for main chain atoms and 2.5 A˚2 for side chain atoms. The model includes residues 6–328, 1 ADP molecule for each of the HP0525 molecules in the asymmetric unit, 1 PEG molecule (n ⫽ 9), and 300 water molecules. All φ, ⌿ angles lie in the allowed region of the Ramachandran plot with 90% in the most favored regions. Acknowledgments We thank J. Vogel, D. Berg, S. Beverley, F. Sauer, C. Frieden, and S. Hultgren for comments on the manuscript and the staff of the Structural Biology Center for assistance during data collection. This work was supported by funds from the Washington University School of Medicine. Received September 13, 2000; revised October 31, 2000. References Abrahams, J.P., and Leslie, A.G.W. (1996). Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D52, 32–42. Asahi, M., Azuma, T., Ito, S., Ito, Y., Suto, H., Nagai, Y., Tsubokawa, M., Tohyama, Y., Maeda, S., Omata, M., et al. (2000). Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J. Exp. Med. 191, 593–602. Backert, S., Ziska, E., Brinkmann, V., Zimny-Arndt, U., Fauconnier, A., Jungblut, P.R., Naumann, M., and Meyer, T.M. (2000). Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus. Cell. Microbiol. 2, 155–164. Bru¨nger, A.T. (1992). The free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–474.

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