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J. Biol. Chem., Vol. 281, Issue 1, 508-517, January 6, 2006
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1
¶2
From the
¶Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada, the Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Western Road, Cork, Ireland, and Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand
Received for publication, July 5, 2005 , and in revised form, October 26, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-helix upon interaction with FliH, and that residues 21–91 of FliI resemble the N-terminal oligomerization domain of the F1-ATPase catalytic subunits. A truncated FliI-(2–91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1–18 of FliI were essential for the FliI/FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal -helix of the F1-ATPase -subunit and the globular domain of the F1-ATPase 
-subunit, respectively. This similarity suggests that FliH may function as a molecular stator. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flagellum secretion machines (7, 13–15) contain a highly conserved flagellum-specific ATPase, FliI, essential for the secretion of flagellar hook and filament proteins. FliI and other export apparatus proteins are thought to associate with the flagellar basal body at the cell membrane (7, 16–23). FliI from Salmonella is a 456-amino acid polypeptide that peripherally associates with the bacterial cytoplasmic membrane (22, 23). In the presence of ATP, Salmonella FliI forms hexamers and demonstrates positive cooperativity of ATP hydrolysis in vitro, and both activities are stimulated in the presence of anionic phospholipids (23). Salmonella FliI has also been shown to interact with FliH, a conserved flagellar export component that inhibits FliI enzymatic activity in vitro (24–28).
Several studies (16–18, 26) suggest that Salmonella FliI contains two domains, a poorly characterized N-terminal segment (amino acids 1–97) with secretion-specific functions and a C-terminal catalytic domain (amino acids 100–456) homologous to the catalytic domains of the - and 
-subunits of F1-ATPase. Mutation of active site residues in the C-terminal domain of FliI are dominant negative for swarming motility (16–18). Similar studies on the homologous InvC type III ATPase also demonstrated negative dominance of catalytic site mutations that were relieved on disruption of membrane localization of the ATPase (20, 21).
The interaction of FliI with FliH, a conserved component of the flagellar and type III secretion systems that inhibits FliI ATPase activity in vitro, has been studied in Salmonella (24–27). An N-terminal FliI double mutant (R7C/L12P) was isolated from genetic screens that detected loss of swarming motility in vivo and acted in a recessive manner (26). This double mutant also failed to interact with FliH in vitro (26), suggesting individual residues in this segment of FliI were required for interaction with one or more components of the export apparatus, including FliH. However, further experiments were not carried out to verify and substantiate this preliminary observation. Furthermore, the extreme nature of the R7C/L12P double mutant suggested this mutation very likely prevented the FliI N terminus from folding into a structure capable of interacting with FliH. Nevertheless, truncated versions of Salmonella FliI purified from limited proteolysis experiments and containing residues 7–456 or 26–456 did not interact with FliH as judged by gel filtration chromatography (24), indicating the N terminus of FliI is largely responsible for interactions with FliH. These studies also indicated that Salmonella FliI was sensitive to clostripain proteolysis at amino acids 7, 26, 93, and 97, but the extent of FliI proteolysis was reduced in the presence of Salmonella FliH (24). FliH itself was not sensitive to clostripain proteolysis.
The domain structure of Salmonella FliH has been studied in some detail. Salmonella FliH forms an elongated dimeric structure in solution and a (FliH)2FliI complex forms with intact full-length FliI (27). A scanning deletion analysis of Salmonella FliH showed that residues 100–235 were required for interactions with FliI; residues 101–141 were required for FliH dimerization (likely via a coiled-coil segment); residues 70–100 were important for inhibition of FliI ATPase activity, and N-terminal residues contributed to binding to the flagellar chaperone FliJ (28). However, these studies do not suggest how FliH inhibits FliI ATPase activity, or what is the general role of FliH in flagellar protein secretion.
Our laboratories are studying flagellar biogenesis in the human gastric pathogen H. pylori as a model for the assembly of a polar sheathed flagellum (10, 19, 29, 30). Thus, we have recently identified the hook length control protein FliK (29) and a novel component that interacts with RpoN and FliH that is essential for flagellum assembly (30). We are also investigating individual flagellar components. The principal objective of this study was to extend our understanding of FliI structure and function based on analysis of the poorly characterized yet important N-terminal region of the molecule (residues 1–91 of H. pylori FliI). Furthermore, we hoped to gain insight into FliI function from analyzing the molecular details of the FliI/FliH interaction. A previously published Y2H partial interaction map of the H. pylori proteome demonstrated that a bait domain containing residues 1–258 of H. pylori FliH interacted with a prey domain containing residues 3–134 of H. pylori FliI (see Ref. 12 and pimhybrigenics.com). However, these studies did not indicate what residues in this fragment of H. pylori FliI were responsible for the interaction with FliH and, as in the Salmonella studies, did not indicate the molecular nature or absolute residue requirements of the interaction. Hence, biochemical confirmation and elaboration of the H. pylori FliI/FliH Y2H interaction (12) were necessary. In addition, no obvious sequence similarity exists between amino acids 1–25 of Salmonella FliI (reported to be required for FliH interaction) and the same region of H. pylori FliI or other FliI sequences.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and -subunit sequences uncovered with the PSI-BLAST search were then prepared as a multiple sequence alignments with T-Coffee (supplemental Fig. S1) (33). In addition, three-dimensional structures are available for the bovine mitochondrial and Bacillus PS3 F1-ATPase - and -subunit sequences (34–36). Therefore, alignment of the FliI/type III ATPase N-domains was verified by using the superimposed structures of the four homologous F1-ATPase subunit N-domains extracted from the Protein Data Bank (www.rcsb.org) as calculated by LSQKAB in the CCP4 suite of programs (37). The most significant alignment to a known structure was to the -subunit of Bacillus PS3 F1-ATPase (25.7% pairwise identity). The structures were visualized on a graphics work station using Turbo-Frodo (38). An homology model of FliI was constructed using Turbo-Frodo, based on the derived alignment and using the F1-ATPase subunit atomic coordinates as templates. The resultant FliI atomic model was energy-minimized using CNS (39). For amino acids 1–18 of FliI sequences given in Fig. S1, secondary structure was predicted using the PHD algorithm as implemented at the Predict-Protein server (40) (cubic.bioch.columbia.edu/predictprotein).
Bacteria and Recombinant Plasmids—DNA manipulation and transformation of E. coli DH5 or XL1-Blue was carried out as described by Sambrook and Russell (41). Gene fragments corresponding to FliH-(2–258), FliH-(55–258), FliH-(94–258), FliH-(117–258), FliI-(2–91), and FliI-(19–91) were isolated by PCR amplification of H. pylori 26695 chromosomal DNA (1–5 ng) by using the appropriate oligodeoxynucleotide primers (supplemental Table S1) (200 nm) in Pfu buffer (Fermentas), 0.2 mM of each dNTP, and 1 unit of Pfu DNA polymerase (Fermentas). Thermal cycling of the PCR was performed in a Bio-Rad MyCycler. Amplified DNA was purified with a QIAquick gel extraction kit (Qiagen), digested with BamHI and EcoRI, and ligated to BamHI/EcoRI-digested pGEX-6P3 glutathione S-transferase (GST)3 fusion protein expression vector (Amersham Biosciences). This vector contains a cleavage site for human rhinovirus 3C protease (PreScission Protease) between the GST tag and the insert. All recombinant proteins described in this study contain an additional N-terminal GPLGS sequence from the BamHI restriction site used in directional cloning of the gene fragments. Plasmids pGEX-6P3-FliI-(2–91) containing N-terminal point mutations were created using 5' primers with a single codon mismatch (see supplemental Table S1). Plasmids were purified from E. coli by using the Qiaprep Miniprep kit (Qiagen). Automated DNA sequencing was used to confirm either the correct DNA sequence or the presence of mutations deliberately introduced by PCR. Primers used in this study are listed in supplemental Table S1.
Overproduction and Purification of N-terminally GST-tagged Proteins—GST fusion proteins containing the truncated proteins FliH-(2–258), FliH-(55–258), FliH-(94–258), FliH-(117–258), FliI-(19–91) FliI-(2–91), and the eight FliI-(2–91) point mutants were purified from the soluble fractions of E. coli Rosetta cells according to the guidelines from Amersham Biosciences for the purification of GST fusion proteins. In brief, the cells were grown at 37 °C to an A600 between 0.4 and 0.6 and induced with 0.1 mM isopropyl -D-thiogalactoside. Following induction, the cells were grown at 25 °C overnight. Cells were harvested, frozen overnight, and lysed by a French press. The supernatant was clarified by centrifuging at 13,000 x g twice for 30 min and then incubated with glutathione-Sepharose for 3 h at 25 °C. The GST affinity tag was removed by adding PreScission protease to the glutathione-Sepharose-bound fusion protein or by adding protease once reduced glutathione was used to release the fusion protein from the resin. For the FliH proteins, they were further purified by anion exchange chromatography on Source Q resin at pH 8.5 followed by gel filtration chromatography in phosphate-buffered saline (PBS) buffer using a Superdex 200 analytical gel filtration column. FliI-(19–91) was purified on Source S followed by gel filtration on Superdex 75. FliI-(2–91) and mutants thereof were purified by glutathione-Sepharose affinity chromatography followed by hydrophobic interaction chromatography (HiTrap phenyl-HP) and finally gel filtration chromatography on Superdex 75.
Molecular Mass Estimation of Purified Proteins—Analytical gel filtration chromatography was used to estimate the molecular masses and respective oligomerization states of purified truncated FliI and FliH proteins. Analytical grade Superdex 75 and Superdex 200 columns from Amersham Biosciences were calibrated with a set of molecular mass standards. The calibration curve plotted is as follows: Kav = (Ve - V0)/(Vt - V0) versus the log10(molecular mass), where Ve is the sample elution volume; Vt is the total bed volume; and V0 is the column void volume. A line of best fit was fitted against the data points.
The Superdex 75 column had a void volume of 7.84 ml determined from the elution of blue dextran, and the total bed volume was 24 ml. The Superdex 75 column was calibrated with the following standards: albumin (Mr = 67,000, Ve = 9.84 ml), ovalbumin (Mr = 43,000, Ve = 10.88 ml), chymotrypsinogen A (Mr = 25,000, Ve = 12.80 ml), ribonuclease A (Mr = 13,700, Ve = 13.80 ml), aprotinin (Mr = 6,500, Ve = 15.73 ml), and vitamin B12 (Mr = 1355, Ve = 19.15 ml). The Superdex 200 column had a void volume of 8.04 ml determined from the elution of blue dextran, and the total bed volume was 24 ml. The Superdex 200 column was calibrated with the following standards: ferritin (Mr 440,000, Ve = 9.12 ml), catalase (Mr = 232,000, Ve = 10.64 ml), aldolase (Mr = 158,0000, Ve = 12.40 ml), albumin (Mr = 67,000, Ve = 14.00 ml), ovalbumin (Mr = 43,000, Ve = 15.12 ml), chymotrypsinogen A (Mr = 25,000, Ve = 16.80 ml), and aprotinin (Mr = 6,500, Ve = 19.01 ml).
GST Pulldown Assays—30 µl of glutathione-Sepharose was prepared by applying four washes of 3 volumes of PBS buffer and then mixing with 20 µg of GST-FliH-(2–258), GST-FliH-(55–258), GST-FliH-(94–258), GST-FliH-(117–258), or GST (all in PBS) for 30 min at room temperature. The protein-bound glutathione-Sepharose was then washed three times with 3 volumes of PBS. Then 4.8 µg of the purified FliI-(19–91), FliI-(2–91), or mutant FliI-(2–91) proteins was added to yield a 2:1 molar ratio of GST-FliH to FliI. The total volume was made to 200 µl with PBS. This mixture was allowed to incubate with gentle agitation for 30 min. The protein-bound glutathione-Sepharose was washed twice with 200 µl of PBS, separated by centrifugation, and boiled with SDS-PAGE loading buffer. The proteins were visualized with Coomassie Brilliant Blue on 15% SDS-polyacrylamide gels. Each well of the pulldown gel contained 20 µg of GST-FliH and 4.8 µg of truncated N-FliI (residues 2–91 or 19–91). Each gel series was repeated in triplicate and shown to be reproducible. Control pulldowns for all of the mutant proteins were conducted but are only shown for GST-FliH-(55–258) (bait) and FliI-(2–91) (prey) (Fig. 5). After GST tag removal, none of the purified prey proteins bound significantly to glutathione-Sepharose. Equivalent protein concentrations were evaluated by measuring protein UV absorbance at 276 nm in PBS buffer for each of the purified proteins based on their aromatic amino acid content.
Far-UV Circular Dichroism Spectroscopy—Far-UV circular dichroism spectra were recorded on a Pistar 180 spectrometer from Applied Photophysics. Spectra were recorded from 180 to 260 nm under a nitrogen atmosphere, and typically a total of 5 scans were recorded at 0.5 nm steps with entry and exit slits set at 4 nm. These scans were then averaged and smoothed to produce the spectra shown. Spectra were measured in 0.1-mm quartz cuvettes at a protein concentration of 0.5 mg/ml in PBS buffer.
Limited Proteolysis of Proteins—Trypsin or chymotrypsin was dissolved to a concentration of 0.1 mg/ml in 10% glycerol and PBS. Protease was added to 50 µg of the protein of interest (FliI-(2–91) or FliH-(2–258)) at a protease to target molar ratio of 1:1000, and the reaction was allowed to proceed for 60 min at room temperature. 10 µg of protein were removed after 5, 10, 20, 40, or 60 min and quenched with phenylmethylsulfonyl fluoride. The protein was visualized with Coomassie Brilliant Blue on SDS-polyacrylamide gels. Bands corresponding to protease digestion products were electroblotted onto polydivinylidine fluoride membranes and then subjected to micro-sequencing. We typically sequenced at least five residues to determine the position of protease cleavage. We also micro-sequenced several of the purified proteins used in this study to verify that their N termini were intact.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit, indicating that the catalytic segments of these proteins descended from a common ancestor, and have a similar three-dimensional structure (16–18, 21). However, the N-terminal domains of both FliI and the F1-ATPase catalytic subunits are also similar in size (
100 amino acids), and this prompted us to ascertain if the evolutionary relationship between these proteins extends to their respective N-terminal domains. PSI-BLAST searches (31, 32) of the nonredundant protein sequence data base demonstrated that residues 20–90 of H. pylori FliI are homologous to the oligomerization domain found in both the - and -subunits of F1-ATPase (E-value scores ranging from 10-165 to 10-135). The alignments span residues 20–430 of FliI and residues 20–430 of either the - or -subunits of F1-ATPase. These results are summarized in a multiple sequence alignment containing FliI and type III ATPase sequences and F1-ATPase - and -subunit sequences whose three-dimensional structures are known (34, 36) (supplemental Fig. S1). There are several invariant glycine residues in the alignment and a number of well conserved hydrophobic residues. The alignment was checked by building a model of residues 20–91 of H. pylori FliI using the structure of Bacillus PS3 F1-ATPase as the template (Fig. 1). The modeled structure indicated the presence of a conserved hydrophobic core in the predicted -barrel domain and that this domain is easily assembled into a hexameric ring structure based on the hexameric arrangement of the F1-ATPase subunit oligomerization domains in the F1-ATPase structure (34, 36). Independent secondary structure predictions of residues 20–90 of FliI (results not shown) also substantiate a -sheet structure for this segment. We also calculated the electrostatic surface potential of the model coordinates for residues 19–91 of hexameric H. pylori FliI (Fig. 1) because the FliI N-domain should be capable of interacting with anionic membrane phospholipids (21–23). The modeled structure does indeed exhibit an electropositive surface potential on the surface most likely to contact the cell membrane (Fig. 1). Hence, we concluded that our alignment and structural modeling for residues 20–90 of FliI reliably predicts a -barrel structure for the N-domain of FliI.
Residues 1–18 of H. pylori FliI Likely Form an Amphipathic -Helix—Residues 1–18 of H. pylori FliI are poorly conserved at the amino acid sequence level when compared with other FliI N-terminal sequences (supplemental Fig. S1) but do have a similar composition enriched in nonpolar and positively charged amino acids. Significantly, hydrophobic amino acids in the N-terminal segment of FliI have a similar periodicity to hydrophobic residues in the N-terminal segment of the F1-ATPase -subunit that is known to form an amphipathic -helix (supplemental Fig. S1) (42). The amphipathic nature of the FliI N-terminal segment suggests this segment is likely to form a short amphipathic -helix (Fig. 1D). Secondary structure prediction indicated that residues 3–12 of H. pylori FliI have a high likelihood of forming an amphipathic -helix. Other FliI N-terminal sequences also exhibit a high probability of helix formation (70–80%). Helical wheel plots of FliI N-terminal sequences, including those of H. pylori and Salmonella (shown in Fig. 1D), are highly suggestive of an amphipathic -helix. These data demonstrate that the FliI N-terminal sequences are consistent with an amphipathic -helical structure. The hydrophilic face of the predicted FliI N-terminal helix would exhibit a positive electrostatic surface, enabling it to potentially interact with anionic membrane lipids. Residues 13–18 of H. pylori FliI likely form a flexible loop, connecting the N-terminal helix to the predicted -barrel domain.
Expression and Characterization of FliI-(2–91) and FliI-(19–91)—To study the N-terminal domain structure of H. pylori FliI and to verify yeast two-hybrid interactions with FliH (12), we cloned a gene fragment corresponding to residues 2–91 of FliI (HP1420) from H. pylori 26695 genomic DNA as a GST fusion construct. The cloning and expression of truncated FliI N-terminal domains has not been attempted previously. GST fusion constructs of FliI-(2–91) purified from E. coli lysates were soluble and were purified to homogeneity (Fig. 2). The purified N-domain of FliI was deduced to be mostly folded from limited proteolysis experiments (Fig. 2) conducted in the presence of trypsin, although residues 2–18 were easily removed by trypsin treatment, as subsequent protein microsequencing of the major proteolysis product revealed a single cleavage site before Arg-19. In addition, the 1H-15N heteronuclear single quantum coherence (HSQC) NMR spectra of a 15N isotopically labeled FliI-(2–91) fragment4 demonstrated good chemical shift dispersion for the majority of the backbone amide N-H peaks. However, 20 of the amide N-H peaks exhibited a narrow chemical shift distribution characteristic of a random coil structure. Far-UV circular dichroism analysis of FliI-(2–91) also indicated a considerable degree of folded structure (Fig. 2). But far-UV CD spectra of a synthetic peptide containing residues 2–14 of H. pylori FliI indicated a random coil structure.5 Therefore, we proposed that residues 1–18 of H. pylori FliI likely correspond to the 20 random coil peaks in the 15N-1H HSQC spectrum and hence are unstructured in solution.
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Because FliI-(2–91) was sensitive to limited proteolysis, we concluded that we could express a discrete domain of FliI containing residues 19–91, and this was verified by cloning, protein expression, and purification (Fig. 2). Again, we estimated the molecular mass of the FliI-(19–91) species by using both Superdex 200 and Superdex 75 analytical gel filtration media (Figs. 2C and 7B). In each case, FliI-(19–91) eluted as a single peak with a predicted molecular mass of 30 kDa. The calculated molecular mass of FliI-(19–91) is 8.3 kDa, and hence we concluded that FliI-(19–91) is also most likely a trimer in solution. The FliI-(19–91) domain was later used as a control in verifying interactions between FliI and FliH.
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-helix (Fig. 3B). However, H. pylori FliH-(117–258) had less helical content than FliH-(94–258) according to the magnitude of the trough at 215 nm in the CD spectra (Fig. 3B). In addition, the gel filtration elution volumes of the truncated FliH proteins were consistent with FliH molecular masses of 58 or 45 kDa, respectively, based on calibration of the column with molecular standards of known mass (Fig. 3A; see "Materials and Methods"). The molecular masses of the FliH-(94–258) and FliH-(117–258) molecules derived from the amino acid sequences are 18.9 and 16.2 kDa, respectively. Hence, each of the truncated FliH molecules could exist either as trimers (56.7 and 48.6 kDa, respectively) or dimers (37.8 and 32.4 kDa, respectively). Although our gel filtration profile data taken alone are most consistent with a trimer of FliH molecules existing in solution, previous studies have shown that Salmonella FliH has a highly asymmetric dimeric structure and hence elutes on gel filtration media at volumes much smaller than those predicted by its apparent molecular mass and known dimeric structure (24, 26, 27). However, the truncated H. pylori FliH molecules used in this study are significantly smaller than Salmonella FliH (141 and 164 amino acids versus 235 amino acids) and may be considerably less aspherical in solution. We tentatively concluded that the solution structure of truncated H. pylori FliH is most likely an elongated dimer that migrates as an anomalously large species on gel filtration media (see "Discussion").
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We then investigated the FliI N-terminal sequence of 18 residues that allowed FliI-(2–91) to interact specifically with FliH. Because we have predicted that this segment of FliI is capable of forming an amphipathic -helix, we hypothesized that a hydrophobic surface from such an FliI N-terminal helix could be the basis for interactions with FliH. We tested this by mutating most of the hydrophobic and positively charged amino acids to alanine or glutamate in residues 2–10 of H. pylori FliI. The proteins corresponding to these eight mutations were then expressed, purified, characterized, and tested for interactions with recombinant FliH (Fig. 5). GST pulldowns were conducted by using equimolar ratios of GST-FliH and each of the respective purified FliI-(2–91) mutant proteins. The GST pulldowns were highly reproducible, and the FliI-(2–91) mutants were then tested with each of the truncated FliH-(55–258), FliH-(94–258), and FliH-(117–258) proteins. All three truncated FliH proteins yielded essentially identical results with the purified FliI-(2–91) mutant proteins (FliH-(55–258) and FliH-(94–258) are shown in Fig. 5). Mutation of Leu-3 to Ala (L3A), Leu-6 to Ala (L6A), or Leu-10 to Ala (L10A) in FliI-(2–91) resulted in a dramatically weakened FliI-(2–91)/FliH interaction. A more drastic change of Leu-6 to Glu (L6E) also disrupted the interaction with FliH and was reproducibly weaker than the Leu to Ala mutations (Fig. 5). Additionally, we made Arg-9 to Ala (R9A), Arg-9 to Glu (R9E), Lys-7 to Ala (K7A), and Lys-4 to Ala (K4A) mutations and tested these for interaction with FliH. R9A, K7A, and K4A mutations interacted as well as wild type FliI-(2–91) with the recombinant FliH proteins (Fig. 5). However, a more drastic Arg-9 to Glu (R9E) mutation significantly weakened the FliI/FliH interaction. Hence, FliI principally interacts with FliH via three leucine residues at positions 3, 6, and 10 in the FliI sequence.
To ensure that the mutant FliI proteins had similar physical and chemical properties to the wild type FliI-(2–91), we examined each of the purified FliI mutants for their oligomerization properties as measured by elution profiles on a Superdex 75 analytical gel filtration column (Fig. 2B). The elution profiles of wild type FliI-(2–91) and the eight tested FliI N-terminal point mutants indicated that the mutant proteins behaved essentially the same as wild type FliI-(2–91) and eluted as a mixture of two peaks (assumed to be trimer and some monomer), although the relative ratio of the two peaks depended on the concentration of loaded protein. Far-UV CD spectra of the FliI-(2–91) point mutants were also indistinguishable from wild type FliI-(2–91).5 Hence, we concluded that the FliI N-terminal point mutations did not appreciably alter the structure of the FliI-(2–91) protein.
We then demonstrated that the FliI-(2–91)-FliH-(117–258) complex is very stable in solution and can be isolated and purified by gel filtration chromatography (Fig. 6). The complex appears to have a 1:1 ratio of FliI-(2–91) and FliH-(117–258) by SDS-PAGE analysis of the peak associated with the eluted complex (Fig. 6). The ratio of proteins visualized on the gel was verified by Coomassie staining of SDS gels of mixtures of known quantities of FliH and FliI (not shown). However, the elution volume of the FliH-FliI complex on Superdex 200 suggests a molecular mass of 1.3 x 105 Da, indicating that more than one copy of FliI-(2–91) and FliH-(117–258) is in the complex. Hence, the stoichiometry of the FliI-FliH complex appears to contain two FliH dimers along with three copies of FliI-(2–91), but this will have to be verified with other experimental approaches. The gel filtration peak associated with the FliI-FliH complex was collected, concentrated, and re-injected onto the Superdex 200 column and showed negligible dissociation into FliI-(2–91) and FliH-(117–258) during elution, indicating that this molecular association is very stable (Fig. 6). This complex does not form when the FliI-(2–91) L3A mutant is incubated with FliH-(117–258) or when FliI-(19–91) is incubated with FliH-(117–258) (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-barrel domain found in the F1-ATPase catalytic subunits. Far-UV CD spectral analysis confirmed that residues 2–91 of H. pylori FliI adopt a mostly folded structure in solution that is absent of significant helical content, consistent with our structural model. Structural modeling further suggests residues 20–91 of FliI may participate in the recognition and binding of anionic phospholipids (Fig. 1). Published studies on the InvC type III ATPase demonstrate that amino acids within the InvC N-terminal domain interact with membrane lipids (21).
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- and -subunits makes important subunit/subunit interactions and exhibits pseudo-hexameric symmetry in the functional F1-ATPase ()3 heterotrimer (34–36). The oligomerization properties of the H. pylori N-domain are in contrast with studies of full-length Salmonella FliI that indicate full-length FliI is largely a monomer in solution, except when ATP or anionic phospholipids are present (23, 27). Full-length H. pylori FliI is also monomeric in solution.5 Therefore, subunit/subunit interactions involving the truncated N-domain of FliI may behave differently in the context of the full-length FliI structure. In other words, the catalytic domain of FliI may impose structural constraints on subunit/subunit interactions involving the FliI N-domain, and these structural constraints may be sensitive to ATP binding in the catalytic domain.
Our work demonstrates that residues 1–18 of H. pylori FliI, although appearing unstructured in solution, are nevertheless absolutely required for interaction with FliH. That this segment likely forms an amphipathic -helix upon interaction with FliH is supported by secondary structure predictions, helical wheel analysis, and mutagenesis results in combination with FliH binding studies. By using site-specific mutagenesis, we showed that three hydrophobic residues in the N-terminal FliI segment (leucine residues 3, 6, and 10) are absolutely required for the interaction with FliH. In contrast, most of the polar residues in this segment appear to have little effect on FliI-FliH complex formation. Hence, we predict that an FliI N-terminal amphipathic -helix forms upon interaction with FliH and that a hydrophobic patch on this helix is critical for productive interactions with FliH. However, there is also a likely electrostatic component to the FliI/FliH interaction as mutation of Arg-9 to Ala did not noticeably affect the stability of the FliI-FliH complex, but the more drastic Arg-9 to Glu mutation significantly weakened complex formation.
Our studies provide significant new insight into to the FliI/FliH interaction. First, although work on the Salmonella FliI/FliH interaction implicated the N terminus of FliI, the experiments either involved use of a drastic FliI R7C/L12P double mutant or used two products purified from limited proteolysis experiments (residues 7–456 and 26–456) (24, 26). Our work demonstrates exactly what residues are important for the interaction in H. pylori FliI (a hydrophobic patch of leucine residues) by using scanning alanine mutagenesis of carefully characterized truncated domains of FliI in combination with a number of truncated recombinant FliH proteins. Incidentally, our results predict that residues 5–12 (5LTRWLTAL12) (hydropholic residues likely to interact with EliH indicated in bold) of Salmonella FliI likely make important contributions to the interaction with FliH (Fig. 1). We conclude that deletion of residues 1–7 of Salmonella FliI (24) likely impaired folding of residues 8–12 of that protein into a helical structure. This potentially also explains why the R7C/L12P double mutant does not interact with FliH (26). Furthermore, our work verifies and significantly extends preliminary yeast two-hybrid data reported for the H. pylori FliI/FliH interaction (12).
The FliI/FliH interaction was further analyzed by isolation and purification of the stable FliI-FliH complex by using gel filtration chromatography on mixtures of purified recombinant FliI-(2–91) and FliH-(117–258). The stoichiometry of the complex as determined by SDS-PAGE indicates an 1:1 molar ratio of FliI-(2–91) and FliH-(117–258). The apparent molecular mass of this FliI-(2–91)-FliH-(117–258) complex is 1.3 x 105 Da, indicating the subunit stoichiometry in the complex is different from the (FliH)2FliI stoichiometry reported for the full-length Salmonella FliI-FliH complex (24). Hence it appears that the N-domain of H. pylori FliI may also behave differently upon interacting with FliH. The functional significance of the FliI-(2–91)-FliH-(117–258) complex is presently unclear.
Data presented in this paper strongly support the contention that residues 1–14 of FliI form an amphipathic -helix upon interaction with FliH. Most intriguingly, the related F1-ATPase -subunit has been shown to contain an amphipathic -helix at its N terminus, just preceding the -barrel domain (supplemental Fig. S1) (42). Furthermore, it is compelling that the F1-ATPase -subunit uses the hydrophobic surface of this -helix to facilitate protein/protein interactions with the F1-ATPase -subunit (42). With FliI, we have shown that hydrophobic residues on this presumed N-terminal helix mediate interactions with FliH. The F1-ATPase -subunit, in combination with the F0F1 b-subunit, is known to function as the stator of the F0F1-ATPase rotary motor, forming an elongated "outer stalk" and preventing unwanted rotation of the F1 catalytic subunits relative to the rotation of the torque-generating 
-subunit (35, 42). We note that the b-subunit of F1-ATPase forms a highly elongated dimeric structure, again reminiscent of the elongated dimeric solution structure of Salmonella and presumably H. pylori FliH. The actual biological function of FliH in flagellar protein export is not known, although in vitro it acts as an inhibitor of FliI catalytic activity (26) and is also known to interact with membrane-embedded components of the flagellar export apparatus and anionic phospholipids (22, 25). Consistent with our observation of similarity in solution properties between FliH and the F0 F1-ATPase b-subunit, other authors have previously noted weak sequence similarity between a segment of the F0F1 b-subunit and FliH, suggesting the possibility of an evolutionary relationship between these proteins (43, 44). We suggest that the FliI/FliH interactions demonstrated in this report are analogous to the observed interactions between F1 - and F1 -subunits in the F0F1-ATPase (42). This implies that the C-domain of FliH (like the F1 -subunit) would have a mostly globular structure responsible for interactions with FliI. Bioinformatic data indeed suggest that the C-domain of FliH adopts a globular structure.6
Previously published data support the idea that FliI functions as a hexameric ring structure (23). The nature of the FliI/FliH interaction and its uncanny similarity to the interaction between an amphipathic helix on the F1-ATPase -subunit and a globular domain on the F1-ATPase -subunit lead us to suggest that FliH could function as the FliI stator. The overall structural features of FliH, a mostly helical N terminus and a globular C-domain together making up an elongated structure, are reminiscent of structural features of the F1-ATPase stator. Structural studies of H. pylori FliI and FliH are in progress in our laboratories to further elucidate the structure/function relationships of these proteins.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Fig. S1 and Table S1.
1 Recipient of a Massey University doctoral scholarship.
2 To whom correspondence should be addressed: Dept. of Biochemistry, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, Saskatchewan S7N 5E5, Canada. Tel.: 306-966-4381; Fax: 306-966-4390; E-mail: stan.moore{at}usask.ca.
3 The abbreviations used are: GST, glutathione S-transferase; PBS, phosphate-buffered saline; HSQC, heteronuclear single quantum coherence.
4 H. Iwai, M. Lane, and S. Moore, unpublished data.
5 M. Lane and S. Moore, unpublished results.
6 S. Moore, unpublished results.
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