Abhrajyoti Ghosh

Sophia Hartung

Chris Van Der Does

John A Tainer

Sonja-Verena Albers
email: albers@mpi-marburg.mpg.de

Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding

Keywords: secretion superfamily ATPase, type II/IV secretion ATPase, Sulfolobus acidocaldarius, Type IV pili, T2S/T4P ATPase superfamily, Lipid activation

come

INTRODUCTION

Motility is vitally important for most prokaryotes, and various mechanisms utilized by bacteria and archaea for motility have been described in recent years [START_REF] Bardy | Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae[END_REF]. In bacteria, the two most commonly used mechanisms, movement via flagellar rotation as well as twitching using the extension and retraction of type IV pili (T4P) have been studied extensively [START_REF] Macnab | Type III flagellar protein export and flagellar assembly[END_REF][START_REF] Hansen | Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion[END_REF]. This has led to the identification and characterization of many components required for the assembly and function of these systems. Very little however is known about the assembly of archaeal surface appendages like flagella and pili, although a diverse variety has been identified [START_REF] Albers | Diversity of archaeal type IV pilinlike structures[END_REF][START_REF] Pohlschroder | Archaeal type IV pilus-like structures-evolutionarily conserved prokaryotic surface organelles[END_REF]. In Halobacterium salinarum flagella have been reported to be involved in swimming motility [6][START_REF] Szabo | Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus[END_REF][START_REF] Lewus | Temperature-sensitive motility of Sulfolobus acidocaldarius influences population distribution in extreme environments[END_REF] and shown to be important for swarming on solid surfaces in methanogens and Sulfolobus solfataricus [START_REF] Szabo | Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus[END_REF][START_REF] Lewus | Temperature-sensitive motility of Sulfolobus acidocaldarius influences population distribution in extreme environments[END_REF]. Recent evidence further revealed that flagella in S. solfataricus are involved not only in surface attachment, but also in late phases of bio-film maturation [START_REF] Zolghadr | Appendage-mediated surface adherence of Sulfolobus solfataricus[END_REF][START_REF] Koerdt | Crenarchaeal Biofilm Formation under Extreme Conditions[END_REF][11].

Remarkably, available structural and genetic information indicated that the archaeal flagella are more similar to bacterial T4P than to the bacterial flagella [START_REF] Albers | Diversity of archaeal type IV pilinlike structures[END_REF][12][START_REF] Trachtenberg | The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure[END_REF][START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF]. Genetic analysis in different flagellated archaea recently has been used to identify a genetic locus that encodes an archaeal flagella assembly system (AFAS) [START_REF] Ng | Cell surface structures of archaea[END_REF]. Within different archaea, this genetic locus contains seven to thirteen genes. Usually it contains several flagellin genes (flaA and/or flaB genes) followed by a conserved set of flagella-related protein genes (flaC-flaJ) [START_REF] Chaban | Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis[END_REF]. The genetic locus in Sulfolobales, however, contains apart from the conserved set of flagella-related protein genes only one gene encoding a flagellin. Several components of the archaeal flagella assembly machinery show homology to proteins involved in type IV pilus (T4P) assembly, type II secretion (T2S), and type IV secretion (T4S) systems of Gram negative bacteria [START_REF] Albers | Diversity of archaeal type IV pilinlike structures[END_REF][START_REF] Szabo | Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus[END_REF][START_REF] Albers | Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF][START_REF] Patenge | The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum[END_REF][START_REF] Cohen-Krausz | The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili[END_REF][START_REF] Cohen-Krausz | The flagellar filament structure of the extreme acidothermophile Sulfolobus shibatae B12 suggests that archaeabacterial flagella have a unique and common symmetry and design[END_REF]. T2S systems in bacteria play a role in the secretion of toxins and enzymes, e.g, the cholera toxin of Vibrio cholerae [START_REF] Mudrak | Specificity of the type II secretion systems of enterotoxigenic Escherichia coli and Vibrio cholerae for heat-labile enterotoxin and cholera toxin[END_REF], whereas T4S systems are used for the transport of DNA to either bacterial or eukaryal host cells as exemplified by Agrobacterium tumefaciens [START_REF] Guo | Recruitment of conjugative DNA transfer substrate to Agrobacterium type IV secretion apparatus[END_REF]. T4P are involved in a variety of functions such as twitching motility and cell adhesion which are essential for the pathogenicity of several harmful bacteria [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF]. All three systems have significant structural and functional similarities pointing to a common origin [START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF]. Interestingly, the subunits that are found to be similar in those systems are also present in the AFAS [START_REF] Ghosh | Assembly and function of the archaeal flagellum[END_REF]. These components include pre-flagellin peptidases (e.g. PibD of S. solfataricus) [START_REF] Albers | Diversity of archaeal type IV pilinlike structures[END_REF], FlaJ, that has homology to the polytopic membrane protein found in T4P systems (e.g. PilG of Neisseria) [START_REF] Albers | Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF]25] and FlaI, that has systems [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF][START_REF] Albers | Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus[END_REF][START_REF] Yamagata | Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism[END_REF][START_REF] Planet | Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification[END_REF]. Members of this superfamily contain characteristic conserved motifs such as Walker A, Walker B and P-loop motifs (GX4GK(S/T)), which are involved in nucleotide binding. They also contain the Asp box motif involved in divalent cation binding and the His box motif possibly is involved in stabilization of the nucleotide binding [START_REF] Possot | Molecular characterization of PulE, a protein required for pullulanase secretion[END_REF].

Based on their sequence similarities, secretion ATPases were grouped into different clusters, including clusters for ATPases from T2S systems, T4P systems, and a cluster for archaeal ATPases [START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF]. Although both T2S and T4S are powered by secretion ATPases, the basic mechanism is different for these secretion systems. In T2S system, the ATPase, GspE is involved in assembly of the 'pseudopilin' filament called pseudopilus. In comparison, T4S system ATPases may function as protein translocators by interacting with the secreted protein already folded in the cytoplasm. In T4P assembly systems, two ATPases, PilB and PilT are present and function in assembly and disassembly of the pilus, respectively [START_REF] Chiang | Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU[END_REF][START_REF] Jakovljevic | PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus[END_REF][START_REF] Turner | Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa[END_REF]. Interestingly, the amino acid sequence of FlaI resembles bacterial T2S and T4P type ATPases, suggesting a common evolutionary root for all these secretion ATPases.

The archaeal flagellum is able to move clockwise and counterclockwise, which results in a switch in swimming direction [START_REF] Schlesner | Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus[END_REF]. The question arises how a type IV pilus would be able to move in this fashion, as normally twitching motility is conferred by filament assembly and disassembly and not by any rotational movement. Because FlaI is the only ATPase identified in archaeal flagella operons, it is most likely involved in two functions: first in the assembly process and secondly in the movement leading to motility. Therefore, it is important to understand how this protein can perform both functions.

The specific role of the secretion ATPase in archaeal flagella assembly systems has not been addressed before. It has been shown for Methanococcus maripaludis and Halobacterium salinarum that FlaI deletion mutants are impaired in assembly of their flagella [START_REF] Chaban | Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis[END_REF][START_REF] Patenge | The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum[END_REF]37], but neither have been biochemically characterized. The relation between ATP binding and hydrolysis and flagellum assembly and rotation remains unknown. In the present work, the secretion ATPase FlaI from Sulfolobus acidocaldarius, a thermoacidophilic crenarchaeon that grows optimally at 70-80˚C and pH 2-3, is biochemically and structurally characterized.

MATERIALS AND METHODS

Construction of a FlaI overexpression vector.

To enable expression of non tagged FlaI or FlaI with an N-terminal His-tag, the flaI gene (saci_1173) was amplified by PCR from genomic DNA of Sulfolobus acidocaldarius DSM639 using primer-pairs FlaI713/FlaI714 or FlaI728/FlaI729 and cloned as NdeI/BamHI or EcoRI/HindII fragment into plasmid pETDuet-1(Novagen), yielding plasmids pSVA251 and pSVA263 respectively. For expression analysis, the respective vectors were transformed to Escherichia coli C43 cells containing the RIL Chlplasmid (Strategene) [START_REF] Miroux | Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels[END_REF].

Site-directed mutagenesis of flaI. Mutagenesis of flaI was performed by overlap PCR on vector pSVA263. Mutants were created by changing K 268 within the predicted Walker A motif, D 290 within the Asp-box and E 336 within the Walker B motif to alanines. The resultant plasmids were named pSVA265 (K 268A ), pSVA307 (D 290A ), pSVA308 (E 336A ) and pSVA314 (K 268A , E 336A ). Sequences of the oligonucleotides used for mutagenesis are listed Table 1.

Expression of recombinant proteins in Escherichia coli. Ten milliliters of an overnight culture of E. coli C43 cells containing the RIL plasmid [START_REF] Miroux | Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels[END_REF] and one of the respective over-expression plasmids was used to inoculate 1 liter of Luria-Bertani medium containing ampicillin (100 µg/ml) and chloramphanicol (34 µg/ml). Cells were grown at 37⁰C to an optical density at 600 nm of 0.6 was reached, at which moment 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added. The temperature was reduced to 16⁰C, and growth was continued over-night to reduce inclusion body formation. The cells were collected by centrifugation, resuspended in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 10% glycerol) containing the complete EDTAfree protease inhibitor cocktail (1 tablet/ 50 ml of lysate; Roche, Manheim, Germany), frozen in liquid nitrogen and stored at -80 o C.

Purification of recombinant proteins. Before purification, frozen resuspended cell pellets were thawed on ice. Cells were lysed by sonication with a Sonoplus HD3100 sonicator (Bandelin Sonorex Biotechnique,Germany) using probe HD3100. Cell debris was removed by centrifugation at 6000 x g for 15 min followed by centrifugation at 150,000 x g for 30 min at 4⁰C in a Optima TM MAX-XP ultracentrifuge (rotor MLA 55) (Beckman Coulter, USA) to remove the membranes. The supernatant was incubated at 70⁰C for 20 min in a water bath. The sample was cooled down on ice and precipitated proteins were removed by centrifugation at 150,000 x g for 30 min at 4⁰C in a Optima TM MAX-XP ultracentrifuge (rotor MLA 55) (Beckman Coulter, USA). For purification of His-tagged proteins, the supernatant was applied to a Ni 2+affinity column (Native IMAC) on the Profinia TM protein purification system (BioRad Laboratories, Inc, USA). Bound protein was eluted in lysis buffer containing 500 mM imidazole. The eluted fraction was dialyzed overnight at 4⁰C against 50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 10% glycerol. The dialyzed fraction was loaded on a Superdex 200 10/300 gel filtration column on an AKTA-Purifier system (GE Health Care, Germany), equilibrated with 50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 10% glycerol (flow rate 0.5 ml/min). The untagged FlaI was expressed, sonicated and heat treated, as described above. The protein was then precipitated at room temperature with 20% ammonium sulfate (w/v) for 30 min. The precipitated protein was resuspended in buffer Blue A (50mM Tris-HCl, pH 8.0, 200 mM NaCl, 2 mM EDTA) and loaded on a 5 ml HiTrap BLUE column in the AKTA-Purifier system (GE Health Care, Germany). The column was washed with buffer Blue A and the bound protein was eluted with buffer Blue B (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 2 mM EDTA). The eluted protein was further purified on a 5 ml HiTrap PHENYL column. The protein was eluted using a gradient from buffer Blue B to buffer PHENYL (50 mM Tris-HCl, pH 8.0, 2 mM EDTA). The eluted protein was then concentrated (<2 ml) using an amicon concentrator system (Milipore, USA), and loaded on to a Superose 6 10/300 size-exclusion column pre-equilibrated with PHENYL buffer and eluted with 50 mM HEPES-NaOH buffer, pH 7.5 containing 150 mM NaCl.

Protein purity was monitored by reducing sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Biosafe (BioRad) protein stain. Protein concentration was determined with the Bio Rad protein assay dye-dependent reagent (Bio Rad).

ATPase assay. ATPase activities were measured using the malachite green assay, as described previously [39]. Briefly, purified protein (2 μg) was preheated for 2 min in 100 μl assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl and 5 mM MgCl 2 ). Reactions were initiated by addition of 1 mM ATP, and incubated for 5 min at 70˚C. The reaction was stopped by freezing in liquid nitrogen. The amount of released inorganic phosphate was determined using the malachite green assay. The data were corrected for non-enzymatic ATP hydrolysis. To determine the activity of FlaI as a function of temperature, pH, nucleotides and divalent cations, the assay was performed at different temperatures, in different buffers (50 mM Citrate, pH 3.0-5.5; MES, pH 6.0-6.5; HEPES, pH 7.0-7.5; Tris-Cl, pH 8.0-9.5), with different NTPs (5 mM of ATP, GTP, CTP, and UTP), and in the absence of, or with different divalent cations (5 mM Na-EDTA or 5 mM MnCl 2 or 5 mM CaCl 2 ,) respectively. The ATPase activity was determined as a function of the ATP concentration, and the resulting data were fitted to the Hill equation y = (ax n /(b n + x n )), to obtain the V max (a), the dissociation constant, K 0.5 (b) and the hill coefficient (n). To analyze the effect of tetraether lipids on ATP-hydrolysis by FlaI, total lipids of S. acidocaldarius were extracted, as described previously [40] . The ATPase activity of FlaI (1 µM purified protein) was measured as described above in the presence of different concentrations of isolated lipids (40, 80, 100, 200, 400, and 800 µg).

MANT-ATP binding assay. Binding of MANT-ATP (2'-/3'-O-(N'-Methylanthraniloyl

)adenosine-5'-O-triphosphate) was followed by FRET measurements using a temperature-controlled ISS PC1 spectrofluorometer (ISS Inc., USA). The excitation wavelength was set to 285 and the emission spectrum was recorded from 400 to 500 nm. The slit widths for the excitation and the emission beam were set to 1 nm. MANT-ATP binding affinities were determined by titrating 0.1 μM of protein with increasing concentration of MANT-ATP (0.125 μM -25 μM) at 25˚C in binding buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MnCl 2 ). Fluorescence spectra were corrected for the fluorescence of unbound MANT-ATP, and the resulting fluorescence at 450 nm was fitted to the Hill equation y = (ax n /(b n + x n )), to obtain the ΔF max (a), the dissociation constant, K 0.5 (b) and the hill coefficient (n).

Liposome pull-down assay. The liposome pull-down assay was performed as described previously [START_REF] Selyunin | The assembly of a GTPasekinase signalling complex by a bacterial catalytic scaffold[END_REF]. Briefly, isolated and dried tetraether lipids from S. acidocaldarius were hydrated with liposome-binding buffer (50 mM HEPES, pH 7.5, 150 mM NaCl and 5 mM MgCl 2 ) and vigorously vortexed to ensure dispersion of the various lipids in a Sonoplus HD3100 sonicator (Bandelin Sonorex Biotechnique, Germany) using probe HD3100. Liposomes were collected from the supernatant after centrifugation (2,500 xg for 5 min) and used in subsequent assays. To perform experiments with E. coli polar lipids, a liquid form of polar lipid mixture was purchased from Avanti Polar Lipids, Inc. (Alabama, USA). The polar lipids were dried in a vacuum desiccator for approximately 5 h. Dried lipids were hydrated with liposome-binding buffer (50 mM HEPES, pH 7.5, 150 mM NaCl and 5 mM MgCl 2 ) and treated in a similar manner as tetraether lipids to generate differentially sized liposomes for pulldown assay.

The pull-down assay was performed separately with 1 M of 6xHistagged FlaI, FlaI K268A or FlaI E336A purified protein. The proteins were preincubated with 1 mM ATP at 25ºC for 30 min in the nucleotide binding buffer (50 mM HEPES, pH 7.5, 150 mM NaCl and 5 mM MgCl 2 ) in a total volume of 100 μL. Liposomes were added to a final lipid concentration of 80 μg/ml and reactions were incubated at room temperature for 1 h. By centrifugation at 100,000 x g in a Beckman TLA100.3 rotor in a Optima TM MAX-XP ultracentrifuge (Beckman Coulter, USA) at 4ºC for 30 min, the lipid vesicles were separated from the soluble fraction. The supernatant and pellet were analyzed on a 11% polyacrylamide gel and visualized with Coommassie blue.

Analytical gel-filtration. Purified FlaI was concentrated to 1.0 mg/ml in 50 mM HEPES-NaOH buffer, pH 7.5, 150 mM NaCl by centrifugation in an amicon filter (10 kDa cutoff; Millipore). The sample was then pre-incubated at 50˚C for 10 min followed by incubation at 4⁰C for 6 h with either 1 mM AMP-PNP (Adenylyl-imidodiphosphate) or ADP in the presence of 10 mM MgCl 2 . The sample was loaded on a Superdex 200 10/300 column on the AKTA purifier system as described above. All the fractions were analyzed on SDS-PAGE and immunoblotted using anti-His antibodies (Abcam).

Small angle X-ray scattering (SAXS) analyses. SAXS data were collected at the ALS beamline 12.3.1 (SIBYLS, Lawrence Berkeley National Laboratory, Berkeley, CA) [START_REF] Hura | Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)[END_REF]. The scattering vector is defined as q = 4π sinθ/λ, where 2θ is the scattering angle. Prior to SAXS data collection FlaI wild-type and FlaI E336A were applied to a superose 6 size exclusion column in 20 mM Tris, pH 7.0, 200 mM NaCl and 20 mM Tris pH 7.0, 200 mM NaCl, 1 mM ATP, 2.5 mM MgCl 2 , respectively. Fractions from the column were analyzed directly and after 2-fold and 4-fold concentration of the sample using Corning Spin-X concentrators. All experiments were performed at 20°C, and data were processed as described [START_REF] Putnam | X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution[END_REF]. The data acquired at short and long time exposure (1 and 10 s) were merged for calculations using the entire scattering spectrum. The experimental SAXS data for different protein concentrations were investigated for aggregation using Guinier plots [START_REF] Guinier | Small Angle Scattering of X-rays[END_REF]. The program This approach also provided the maximum dimension of the macromolecule, D max . The overall shapes were restored from the experimental data using the program GASBOR and averaging of 15 independent structures using the program DAMAVER [START_REF] Svergun | Determination of domain structure of proteins from X-ray solution scattering[END_REF]. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081, Pettersen et al, 2004). Theoretical scattering curves from crystal structures were calculated using FoXS [47].

RESULTS

Expression, purification and biochemical characterization of FlaI

Both His-tagged and wt FlaI were expressed in E. coli and purified to homogeneity. Purified FlaI was analyzed by SDS-PAGE, and found to be over 99% pure. FlaI was tested for the ability to hydrolyze ATP under different conditions. The optimal conditions for ATP hydrolysis by FlaI were determined to be pH 6.5 and 75˚C (Figure 1A and1B), which are close to the cytoplasmic pH and the optimal growth temperature of Sulfolobus acidocaldarius. Mn 2+ was the preferred divalent cation for ATP hydrolysis (Figure 1C), as was observed for other secretion ATPases [START_REF] Albers | Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus[END_REF]. Considerable activity was however also detected in the presence of Ca 2+ and Mg 2+ , respectively (Figure 1C). FlaI hydrolyzed ATP with the highest rate, but it was also able to hydrolyze GTP, CTP and UTP (Figure 1D), as seen for other ATPases. No conversion of ADP to AMP was detected (Figure 1D).

To determine the concentration dependency of ATP hydrolysis, the ATP hydrolysis rate was determined as a function of the ATP concentration ( Figure 1E and1F). The fit of the data with the Hill equation resulted in a V max of 32 nmol Pi/mg protein/min, a K 0.5 of 10 mM and a Hill coefficient of 2.0. The Hill coefficient demonstrates that ATP hydrolysis is cooperative, suggesting that FlaI oligomerizes during ATP hydrolysis. FlaI had a very low turn-over rate (1 ATP/min), consistent with observations on other secretion ATPases [START_REF] Albers | Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus[END_REF]. Most of these ATPases are part of large macromolecular assemblies and other protein components of these complexes, for example, the membrane protein EpsL in type II secretion system, stimulate the nucleotide hydrolyzing activity of the ATPases [START_REF] Camberg | Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids[END_REF][49]. Furthermore, lipids can enhance the ATPase activity of these proteins [START_REF] Camberg | Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids[END_REF].

To assess the influence of archaeal tetraether lipids on the FlaI ATPase activity, the specific activity of the enzyme was measured in the presence of increasing concentrations of S. acidocaldarius tetraether lipids (Figure 2). Remarkably, the tetraether lipids increased the ATPase activity 3-4 fold, whereas the addition of an E. coli total lipid extract only led to a 1.5 fold stimulation of the ATPase activity. This is in contrast to EpsE, which activity was only activated in the presence of both lipids and the cytoplasmic loops of the corresponding inner membrane protein and not by lipids alone [START_REF] Camberg | Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids[END_REF].

Active site mutants of FlaI

Secretion ATPases contain key conserved motifs for the Walker A and B motifs and the Asp-box. Alignment of S. acidocaldarius FlaI with GspE, i.e. a secretion ATPase of Archaeoglobus fulgidus [START_REF] Yamagata | Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism[END_REF], led to the identification of these motifs in FlaI (Figure S1). To determine the effects of mutations in these motifs, the lysine of the Walker A (K 268A ), the glutamate of the Walker B (E 336A ), and the aspartate of the Asp-box (D 290A ) were mutated. A double mutant of the Walker A and B mutations FlaI K268A/E336A was also constructed. These mutants were expressed and purified, and the expression levels and final purity of these proteins were equal to wt FlaI (data not shown). The different mutants were tested for their ability to hydrolyze ATP (Figure 3A), and for their ability to bind the fluorescent ATP analog MANT-ATP ( Figure 3B). Remarkably, mutation of the lysine of the Walker A motif to an alanine only resulted in a reduction of approximately 50% of ATP-hydrolysis compared to wild type FlaI, while mutagenesis of the aspartate of the Asp-box had no effect on ATP hydrolysis. By substituting E 336 of the Walker B with an alanine, ATPase activity was reduced upto 90%. Analysis of the crystal structure of GspE from A. fulgidus (pdb: 2OAP) revealed that the glutamate residue (E 336 in FlaI) in the Walker B motif is involved in the interaction with Mg-ATP and also is highly conserved among secretion ATPases superfamily members from various archaeal species (Figure S1) [START_REF] Yamagata | Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism[END_REF]. Also for the Myxococcus xanthus PilT, which is the disassembly ATPase of the T4P assembly system, mutagenesis of the conserved glutamate residue in the Walker B box to alanine resulted in loss of ATPase activity [START_REF] Jakovljevic | PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus[END_REF]. Almost no ATP hydrolysis (2-3%) was detected for the FlaI K268A/E336A double mutant.

The MANT-ATP binding assay demonstrated that wt FlaI bound MANT-ATP with a K d of 0.46 μM, a fit of the data with the Hill equation resulted in a Hill coefficient of 0.9, demonstrating that ATP binding, contrary to ATP hydrolysis was not co-operative. Remarkably, MANT-ATP binding to both the Walker B motif (K d = 0.58 μM) and Asp-box mutant (K d = 0.43 μM) was comparable to wt FlaI, whereas binding to the Walker A mutant was strongly reduced (K d = 9.6 μM). Thus our data showed that mutagenesis of the Asp-box residue of FlaI does not have any effect on ATP binding and hydrolysis, while mutation of the Walker B motif does not affect ATP binding, but heavily abolishes ATP hydrolysis. Notably, mutagenesis of the Walker A motif results in a 20-fold lower binding affinity but an only 2-fold reduction of ATPase hydrolysis activity. Presumably, the Walker A box substitution, i.e. K 268A in FlaI, indirectly reduces ATPase activity by interfering with ATP binding. The Walker B box substitution on the other hand, i.e. E 336A in FlaI, abolishes ATPase activity by directly interfering with the ATP hydrolysis, although ATP binding seems normal in this mutant (E 336A ). Furthermore, the data suggest no role of the conserved D 290 in the Asp-box motif in ATP binding and hydrolysis.

Oligomerization of FlaI

Members of the secretion ATPases superfamily can oligomerize into higher oligomeric structures [START_REF] Yamagata | Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism[END_REF][START_REF] Patrick | Oligomerization of EpsE coordinates residues from multiple subunits to facilitate ATPase activity[END_REF][51]. To assess the oligomeric state of purified FlaI (monomer molecular weight 55 kDa), his-tagged FlaI was analyzed by size exclusion chromatography. Due to the presence of a small shoulder in the chromatogram, small amounts of a dimeric FlaI seem to be present (Figure 4, fraction 13). To determine whether FlaI could form higher oligomeric structures, the effect of nucleotide binding on FlaI oligomerization was studied using the non-hydrolyzable ATP analog AMP-PNP. After preincubation with or without AMP-PNP and Mg 2+ , purified FlaI was analyzed by size exclusion chromatography. Remarkably, after incubation with AMP-PNP, FlaI was eluted at estimated molecular weights of 330 kDa and 55 kDa, respectively. This shows that FlaI assembles in an oligomeric structurein a nucleotide-dependent manner in the presence of non-hydrolyzable ATP analogs. To ascertain that the same phenomenon occurs in FlaI mutants, FlaI with a mutation in either the Walker A motif (FlaI K268A ) or the Walker B motif (FlaI E336A ), which showed a reduced (~50%) and very low (~10%) ATPase activity, respectively, were also analyzed. After incubation with ATP, a higher oligomeric form was observed only for the FlaI protein containing a mutation in the Walker B (FlaI E336A ), but not for the FlaI with a mutation in the Walker A motif (FlaI K268A ) (Figure S2). This demonstrated that FlaI with a mutation in the Walker B motif (FlaI E336A ) forms a stable oligomer after ATP binding. Therefore, wt FlaI and FlaI with a mutation in the Walker B motif (FlaI E336A ) oligomerize upon ATP binding. However, the oligomerization potential of Walker B motif mutant (FlaI E336A ) seems higher compared to wt FlaI, suggesting a more stable complex formation with ATP by the Walker B mutant (FlaI E336A ) (Figure S2). As expected, incubation of either the wt FlaI, or FlaI with a mutation in either the Walker A motif (FlaI K268A ) or the Walker B motif (FlaI E336A ) with ADP did not result in oligomerization of FlaI (data not shown).

SAXS analysis of oligomerization states of FlaI

To further analyze the oligomeric structure of FlaI, Small Angle X-ray Scattering (SAXS) was performed. SAXS is a powerful method to structurally analyze proteins in solution under physiological conditions [START_REF] Putnam | X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution[END_REF]. In Figure 5, the SAXS analysis of wild-type FlaI is visualized in comparison to the Walker B mutant (FlaI E336A ) in the presence of ATP and Mg 2+ . The experimental data was compared with theoretical SAXS curves from the crystal structures of monomeric and hexameric GspE. In order to obtain a reasonable fit of the monomeric GspE to the SAXS data from monomeric FlaI, the N-terminal domain (in orange) had to be extended from the ATPase domain (green), which is an indication that there is a flexible conformation of these domains relative to one another. FlaI E336A with ATP clearly forms a hexameric ring similar to the one determined for GspE.

Ab initio structures for both complexes agree well with the GspE models. For calculating the structure of the hexameric protein, we used both no symmetry constraint and 6-fold symmetry. Even without symmetry constraints, we obtained the closed ring structure, but the conformation of the N-terminal domains (orange) seems to be different for the single subunits. The SAXS data from FlaI in monomeric and hexameric form can be analyzed by the Kratky plot (Figure S3) to determine amounts of intrinsic flexibility. In the monomeric form of FlaI that is present in the cytoplasm, the amount of flexibility within the protein is much higher compared to the hexameric, membrane associated form. From this data, we propose that the N-terminal domains are flexible when FlaI has a cytoplasmic localization but may adopt a more rigid conformation when the proteins attach to the membrane or membrane proteins.

Liposome interaction studies

The structure of GspE showed an accumulation of hydrophobic patches at the interface which probably interacts with membrane-integral components. The same hydrophobicity was observed in FlaI models based on the GspE structures. Based on the 3-4 fold increase in FlaI ATPase activity upon addition of archaeal tetraether lipids, it was essential to ascertain that a direct interaction between lipids and FlaI occurs. To this end, a liposome pull-down assay was performed in which FlaI, FlaI K268A and FlaI E336A were incubated either with S. acidocaldarius tetraether lipid liposomes (Figure 6A) or E. coli lipid liposomes (Figure 6B). In the case of the tetraether lipids, the FlaI association with the liposomes was ATPdependent. When ATP was present, FlaI E336A associated most strongly with the tetraether liposomes whereas FlaI K268A bound the least. The same results were observed when the same experiment was performed in the presence of AMP-PNP (Figure S4).These data imply that ATP binding by FlaI is essential for its interaction with tetraether lipids. In contrast, the interaction of FlaI with the E. coli lipids was independent of the presence or absence of ATP, showing that this is an unspecific interaction that is caused by the hydrophobic nature of the upper surface of the hexameric protein. A c c e p t e d M a n u s c r i p t DISCUSSION Although archaeal flagella systems were identified some time ago, the role that the various flagellar subunits play during assembly and rotation and how they interact with each other during this process has not been elucidated [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF]. The secretion ATPase FlaI is a central component of the flagellum assembly system. Previously, it was shown that FlaI could hydrolyzes ATP under in vitro condition [START_REF] Albers | Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus[END_REF]. Genetic analysis revealed its importance in the assembly process. flaI deletion mutants of M. maripaludis and H. salinarum were impaired in flagella assembly [START_REF] Chaban | Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis[END_REF][START_REF] Patenge | The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum[END_REF]37]. Furthermore, in H. salinarum it has been shown that flagellar rotation is dependent on the intercellular ATP concentration and not on the proton motive force, as is the case in bacteria [START_REF] Streif | Flagellar rotation in the archaeon Halobacterium salinarum depends on ATP[END_REF]. These findings implicate FlaI as an essential ATPase for flagellar assembly and/or rotation. However, to provide further insights into its roles, it is necessary to understand the molecular mechanism that FlaI employs. Here, we demonstrated that FlaI hydrolyzes ATP and undergoes an ATP-dependent oligomerization. Computational analysis has identified the conserved ATP binding (Walker A), metal binding (Asp box), and ATP hydrolysis (Walker B) motifs in FlaI. We demonstrated the importance of each of these predicted domains in ATP binding and hydrolysis by site-directed mutagenesis. ATP-hydrolyzing activity of FlaI is more efficient using Mn 2+ as the co-factor instead of Mg 2+ or Ca 2+ . This preference for Mn 2+ as co-factor is congruent with secretion ATPases and kinases characterized from S. solfataricus [START_REF] Albers | Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus[END_REF]. Our data also revealed that the ATPase activity of FlaI displays positive cooperativity, indicating that interactions between subunits occur in an ATP-dependent manner. However, we found that MANT-ATP binding is non-cooperative, probably because ATP analogs bind the monomeric or dimeric state of FlaI under in vitro conditions. Analytical gel filtration demonstrated that in the presence of a nonhydrolyzable ATP analogue the monomer shifted to a higher oligomer and SAXS confirmed that the higher molecular species was a hexamer. Importantly, archaeal tetraether lipids were found to enhance the ATPase activity of FlaI, indicating that the FlaI transition state intermediate is possibly stabilized in an ATP-bound state. The interactions of the ATPase motor with membrane lipids may therefore help regulate its actions. Our data thus provide the first substantial insights into archaeal FlaI function and probably preface the understanding of a common mechanism of FlaI homologues in the Archaea. Generally, these data suggest a hexameric state of FlaI in its ATP-bound state, when it is involved in flagellum assembly. Collectively, these data provide the basis for a model for the mode of function of FlaI (Figure 7). Under in vivo conditions, FlaI hydrolyses ATP and provides energy to the AFAS. It is still a matter of contention, whether the energy gained in this process is utilized for flagellar assembly by directing the translocation of structural components like flagellins or for the rotational movement of flagella or even for both. Crystal structures of the archaeal secretion ATPase GspE, the H. pylori and E. coli hexameric T4S ATPases, as well as the hexameric PilT ATPase in the T4P assembly system of P. It is possible that ATP hydrolysis is closely coupled to flagellin export into the growing flagellar filament. In contrast to this idea, ATP hydrolysis of FlaI might generate energy for the rotation of the flagellum as it was shown that in H. salinarum flagellar rotation is ATP dependent [START_REF] Streif | Flagellar rotation in the archaeon Halobacterium salinarum depends on ATP[END_REF]. Understanding how components in AFAS function to effect proper assembly on the archaeal surface and also how they communicate to execute these functions are still at an early stage. The secretion ATPase FlaI and polytopic membrane protein FlaJ are potentially involved in formation of the core of the assembly complex, and it has been postulated that on this FlaI-J core is the main platform for flagellar assembly [12,[START_REF] Albers | 5'-GGGGGCTCGAGTCAAATATTACTGACATATTTTATC-3'; Reverse primer for FlaI containing XhoI restriction site 5'-CCCCCGAATTCGATGAGCTTCGTGGAAGAC-3'; Forward primer for FlaI containing EcoRI restriction site 5'-CCCCCAAGCTTTCAAATATTACTGACATATTTTATC-3'; Reverse primer for FlaI containing HindIII restriction site 5'-ATCTGGAGCGACAACTAC-3'; Overlapping internal forward primer for K 268 A mutation in FlaI 5'-AATGTAGTTGTCGCTCCAG-3'; Overlapping internal reverse primer for K 268 A mutation in FlaI 5'-TATTGAGGCGACTCCAG-3'; Overlapping internal forward primer for D 290 A mutation in FlaI 5'-TCTGGAGTCGCCTCAATA-3'; Overlapping internal reverse primer for D 290 A mutation in FlaI 5'-ACTTGTGGGAGCAATTAGAG-3'; Overlapping internal forward primer for E 336 A mutation in FlaI 5'-CTTTGTCTCTAATTGCTCCCACAAG-3'; Overlapping internal reverse primer for E 336 A mutation in FlaI[END_REF]. However, another incomplete ATPase, FlaH has also been postulated to function in association with the FlaIJ core to facilitate the process of assembly, disassembly or rotation. Understanding the biochemical and structural features of the FlaI protein provides a basis to understand the function that this protein fulfills in AFAS which can be transferred to homologous ATPases from other T4P systems of bacteria.

Table 1 Primers and plasmids used for assays

Bacterial strains, primers and plasmids Relevant characteristics

Source or reference

Biochemical

  Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Biochemical Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410 A c c e p t e d M a n u s c r i p t GNOM [45] was used to compute the pair distance distribution functions, P(r).

  Biochemical Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410

  aeruginosa have been shown to use N-terminal domain movement to facilitate closed ATP-bound and open ADP-bound hexameric conformations [27-28, 51, 53-55]. Furthermore, it has been Biochemical Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410 A c c e p t e d M a n u s c r i p t postulated that these ATPases act as inner membrane gated transporters for structural proteins and may thus mediate the whole cycle of assembly by executing a conformational change from closed to open upon ATP hydrolysis.

  lacZDM15 D(lacZYA-argF)U169 recA1 endA1 hsdR17 (rK2 mK1) supE44 thi-1 gyrA relA1 B F-ompT hsdS(rB-mB-) dcm+ Tetr E. coli gal λ (DE3) endA Hte [argU ileY leuW Camr] F-ompT gal hsdSB (rB-mB-) dcm lon _DE3 endA Hte [argU ileY leuW Camr]Hyperthermo-acidophilic crenarchaeon which grows optimally at 80˚C and pH 2Car r , expression plasmid containing replicon ColE1 (pBR322) and two MCS (MCS1 and MCS2) Amp r , Car r , pETDuet-1 carrying untagged FlaI in MCS2 using restriction sites NdeI-XhoI Amp r , Car r , pETDuet-1 carrying N-terminal His-tagged FlaI in MCS1 using restriction sites EcoRI-HindIII Amp r , Car r , pETDuet-1 carrying N-terminal His-tagged FlaI(K 268 A) in MCS1 using restriction sites EcoRI-HindIII Amp r , Car r , pETDuet-1 carrying N-terminal His-tagged FlaI(D 290 A) in MCS1 using restriction sites EcoRI-HindIII Amp r , Car r , pETDuet-1 carrying N-terminal His-tagged FlaI(E 336 A) in MCS1 using restriction sites EcoRI-HindIII Amp r , Car r , pETDuet-1 carrying N-terminal His-tagged FlaI(K 268 A, E 336 A) in MCS1 using restriction sites EcoRIPublication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Figures and figure legends

  Figures and figure legends

Figure 1

 1 Figure 1 ATPase activity of FlaI under various conditions Total Pi released upon ATP (NTP) hydrolysis was measured by the malachite green assay. (A) Influence of pH on FlaI ATPase activity. (B) Influence of temperature on FlaI ATPase activity. (C) Preference for optimal divalent metal ion as cofactor in FlaI ATPase activity. The final concentration of protein used in the assays was 1 µM and the final concentration of metal ions was 5 mM. (D) Analysis of optimal substrate for hydrolysis. NTPs were added to a final concentration of 5 mM. (E) FlaI ATPase activity as a function of the ATP concentration measured at 70°C with 1 µM of FlaI. (F) The same FlaI ATPase

Figure 2

 2 Figure 2 Effect of tetraether lipids from S. acidocaldarius and total lipids from E.coli on FlaI ATPase activityATP hydrolysis was measured at 70°C with 1 µM of purified FlaI in presence of different concentrations (40 µg-800 µg) of isolated tetraether-linked lipids from Sulfolobus acidocaldarius and total lipids from E.coli against respective controls in which an equivalent amount of respective lipids was present in the assay mixture without any protein and treated in a similar manner as for the enzymatic reaction described in materials and methods. All experiments were performed in triplicate and standard deviations are indicated.

Figure 3 .Figure 4 Figure 5

 345 Figure 3 Characterization of ATP-binding and hydrolysis motifs in FlaI (A) Comparative ATP-hydrolyzing activity of Walker A (FlaI K268A ), Asp-box (FlaI D290A ), Walker B (FlaI E336A ), and Walker A & B double (FlaI K268A, E336A ) mutants against the wild type FlaI protein (black). (B) Analysis of the binding of MANT-ATP by wild type FlaI and the mutants as monitored by FRET. The change in fluorescence (ΔF) upon MANT-ATP binding is shown as a function of the concentration of MANT-ATP. Fluorescence spectra were corrected for the fluorescence of unbound MANT-ATP. The K d values were calculated for each of the mutants and wild type FlaI. Closed circles, FlaI; closed triangle, FlaI K268A, E336A : open rectangle, FlaI K268A ; vertical stripe, FlaI D290A ; closed diamonds, FlaI E336A .

Figure 6

 6 Figure 6 Liposome pull-down assay of FlaI in the presence of S. acidocaldarius tetraether lipids and E.coli lipid extractThe interaction of either FlaI, FlaI K268A or FlaI E336A protein was tested either with S. acidocaldarius tetraether liposomes (A) or lipids from E. coli (B) in presence or absence of ATP. After centrifugation, proteins remaining in the supernatant (s) or those associated with liposomes in the pellet (p) were separated on an SDS-PAGE.

Figure 7

 7 Figure 7 Model describing the sequence of events that FlaI undergoes in AFAS as inferred from this study

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

We thank Benjamin Meyer for isolating the archaeal lipids. AG received a Max Planck Postdoctoral fellowship and SVA was supported by a VIDI grant of the Dutch Science Organization (NWO) and intramural funds of the Max Planck Society. SH and JAT T4P system efforts are supported by National Institute of Health [AI022160] and microbial complex SAXS efforts are supported by the ENIGMA Program of the Department of Energy, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 with Lawrence Berkeley National Laboratory and by National Institutes of Health grant AI022160. The SIBYLS beamline (BL12.3.1) at the Advanced Light Source is supported by United States Department of Energy program Integrated Diffraction Analysis Technologies (IDAT) DE-AC02-05CH11231. We thank Michal Hammel for expert SAXS data collection assistance. We thank Andrew Bozarth for critical reading of the manuscript. Biochemical Journal Immediate Publication. Published on 20 Apr 2011 as manuscript BJ20110410 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110410 A c c e p t e d M a n u s c r i p t

This study This study This study This study This study This study This study This study This study This study

Primers