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J. Biol. Chem., Vol. 281, Issue 3, 1532-1546, January 20, 2006
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24
From the
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the ¶Samuel Lunenfeld Research Institute, Mount Sinai Hospital and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the ||Banting and Best Department of Medical Research and Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and the Laboratoire de Virologie Moléculaire et Structurale, UMR 2472/1157, CNRS-Institut National de la Recherche Agronomique and Institut Fédératif de Recherche 115, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
Received for publication, October 13, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S transcription factor, and its levels increase toward late log/early stationary phase, consistent with its possible role as a general stress-response protein. RavA functions as an ATPase and forms hexameric oligomers. Importantly, we demonstrate that RavA interacts strongly with inducible lysine decarboxylase (LdcI or CadA) forming a large cage-like structure consisting of two LdcI decamers linked by a maximum of five RavA oligomers. Surprisingly, the activity of LdcI does not appear to be affected by binding to RavA in a number of in vitro and in vivo assays, however, complex formation results in the stimulation of RavA ATPase activity. Data obtained suggest that the RavA-LdcI interaction may be important for the regulation of RavA activity against its targets. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent phylogenetic analyses using sequence and structural information have identified numerous families of AAA+ proteins (1, 2, 7, 8). One major but poorly characterized family is the MoxR family, which is widespread throughout bacteria and archaea, with members being represented in all major lineages (1, 2, 8). The exact functional role of MoxR proteins is unclear, although limited work with members of this family suggests a chaperone-like role in the assembly/activation of specific protein complexes. The first characterized member of this family is MoxR in Paracoccus denitrificans, which was shown to be important in the biogenesis of methanol dehydrogenase. Cells in which the moxR gene was disrupted produced wild-type levels of methanol dehydrogenase, but, surprisingly, the enzyme was not functional (9). Another MoxR-related protein, NorQ/NirQ, was shown to be important in the biogenesis of nitric-oxide reductase. As with MoxR and methanol dehydrogenase, disruptions of the norQ/nirQ gene resulted in the production of non-functional nitric-oxide reductase enzyme in Pseudomonas stutzeri, P. denitrificans, and Rhodobacter sphaeroides (10–12). Also, the CbbQ gene product, a MoxR-related protein, was shown to alter Rubisco4 conformation and stimulate its activity upon overexpression of recombinant forms of the proteins together in an Escherichia coli system (13, 14). Yet another MoxR family member, GvpN, has been shown to be important in gas vesicle biogenesis in certain organisms (15–18). The exact role and mechanism of function of MoxR proteins in all of these processes are not clear.
Genes encoding MoxR proteins frequently co-occur in close proximity to genes encoding proteins containing Von Willebrand Factor Type A (VWA) domains, suggesting that these gene products may be functionally linked. For example, the MoxR family members, norQ and cbbQ genes, are encoded on the same operon as, and immediately upstream of, the norD and cbbO genes, respectively, which encode VWA proteins. Both NorD and CbbO were shown to be important in the activation of functional nitric-oxide reductase and Rubisco, respectively (11–13). The VWA domain is a metal-binding domain often mediating protein-protein interactions (19). The metal is typically magnesium. Metal-binding occurs through a non-contiguous metal ion-dependent adhesion site, which is important for binding protein ligands (20). VWA proteins have been well studied in eukaryotes, where they are involved in a range of processes, including cell adhesion, transport, the complement system, proteolysis, transcription, DNA repair, and ribosome biogenesis. Bacterial and archaeal VWA proteins are not as well characterized, although limited work has identified members involved in surface adhesion, serum opacity, fibrinogen binding, and metal insertion into protoporphyrin IX (21–23). Thus, to carry out their varied remodeling functions, many MoxR AAA+ proteins probably work in conjunction with VWA domain-containing proteins.
As part of a research project aimed at identifying novel AAA+ chaperones in E. coli, we became interested in the uncharacterized protein YieN (SwissProt number P31473 [GenBank] ), which defines a MoxR subfamily (2). We have renamed this protein RavA (regulatory ATPase variant A). Here, we show that RavA is a cytoplasmic, hexameric AAA+ protein that functions with a VWA domain-containing protein, YieM (SwissProt number P03818), which we termed ViaA (VWA interacting with AAA+ ATPase). Detailed information is presented on RavA gene organization, expression patterns, protein subcellular localization, enzymatic activity, and oligomerization properties. Furthermore, we show that RavA strongly interacts with the inducible lysine decarboxylase (LdcI or CadA, SwissProt number P0A9H3), an enzyme important in bacterial acid stress response, to form a distinctive 5-fold symmetric cage-like complex as determined by electron microscopy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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36 sequences identified as belonging to the RavA subfamily of MoxR AAA+ were selected and aligned using ClustalW. This alignment was then used to identify residues conserved in >90% of sequences. Conservation was based upon the following amino acid groupings: ILVM, KR, DE, ST, YFW, and NQ. Ungrouped amino acids included Ala, Cys, Pro, His, and Gly.
Bacterial Strains—Wild-type MG1655 strain was purchased from ATCC (ATCC Number 47076). MG1655 
ravA strain was constructed from WT MG1655 using the Lambda Red system as described (30, 31). Briefly, knock-out cassette containing the chloramphenicol resistance marker was amplified by PCR from pKD3 plasmid using primers RKO-_forward (5'-agaaacgtctatactcgcaatttacgcagaacttttgacgaaagggtgtaggctggagctgcttc-3') and RKO_reverse (5'-aatctgtgcaccgacatcctgtaggctggcttcaatgcgacctaacatatgaatatcctccttag-3') to construct the ravA deletion strain, or VKO_forward (5'-ctgaaggcggcaatcactgatgatgttccccgctggcgtgaaggtgtaggctggagctgcttc-3') and VKO_reverse (5'-gcgagagcgtcccttctctgctgtaataatttatcgccgccagcgcatatgaatatcctccttag-3') to construct the viaA (yieM_ECOLI) deletion strain. DY330 cells were inoculated from single colonies into 50 ml of LB media and grown at 30 °C with shaking to A600 = 0.6–0.8. 25 ml of culture was heated in a 42 °C water bath with shaking for 15 min to induce expression of the Lambda Red genes exo, bet, and gam. Cells were immediately cooled on ice for 10 min. Cells were made electrocompetent and then transformed with 1–2 µl of linear PCR product using a Bio-Rad GenePulser. Transformed cells were diluted into 1 ml of LB and grown at 30 °C for 1 h. Cells were then plated onto LB agar containing 25 µg/ml chloramphenicol and grown overnight at 30 °C. Resistant colonies were selected and screened for successful deletion of the gene(s) using PCR. The deletion cassette was then transferred from DY330 to MG1655 using P1dam rev6 transduction as described previously (32). Verification of the deletion strains was performed using PCR, DNA sequencing, and Northern and Western blot analyses. It should be noted that deletion of ravA resulted in a strain that also cannot transcribe viaA (see Fig. 4, A and B).
The RavA-TAP-encoding strain was constructed using the Lambda Red system in a manner similar to that described for the construction of the knock-out strains. Specific details on strain construction, as well as on the pull-down and identification of bands by mass spectrometry, are provided elsewhere (33). MG1655 rpoS and cadB strains were obtained from the E. coli genome project at the University of Wisconsin, Madison, WI.
Gene Cloning—ravA (yieN_ECOLI), ldcI (cadA/dcly_ECOLI), and ldcC (dclz_ECOLI) genes were cloned from E. coli K12 MG1655, whereas viaA was cloned from E. coli O157:H7. It should be noted that the amino acid sequences of ViaA from E. coli K12 MG1655 and O157:H7 strains are identical. Primers used were as follows: RAVA_forward (5'-atcacgatcatatggctcaccctcatttattag-3'), RAVA_reverse (5'-tacgtaggatccttagcattgttgtgcctggcg-3'), VIAA_forward (5'-aactcgatcatatgctaacgctggatacg-3'), VIAA_reverse (5'-ctatggatccttatcgccgccagcgtctgagc-3'), LDCI_forward (5'-atgctaccatatgaacgttattgcaatattg-3'), LDCI_ reverse (5'-ctggatccttatttcttgctttcttctttcaatacc-3'), LDCC_forward (5'-tctaccatgggcatgaacatcattgccattatg-3'), and LDCC_reverse (5'-cacctcgagttatcccgccatttttaggac-3').
ravA was cloned into p11 plasmid (34), a modified pET15b, which adds His6 followed by a tobacco etch virus-cut site to the N terminus of the expressed protein (HV tag). viaA and ldcI were cloned into pET3a (Novagen), which expresses untagged proteins. ldcC was cloned into pET16b such that it was expressed untagged, with the exception of additional N-terminal Gly and Met residues. All constructs were verified by DNA sequencing.
Protein Expression and Purification—Proteins were expressed in BL21(DE3) pLysS (Stratagene). RavA, ViaA, and LdcI protein expression was induced by addition of 0.4 mM isopropyl 1-thio-
-D-galactopyranoside to cultures grown to midlog phase. Cultures expressing RavA were grown overnight at 30 °C in the presence of inducer, and those expressing ViaA were induced at 18 °C overnight. Cultures expressing LdcI were induced for 3 h at 37 °C. LdcC cultures were grown at 37 °C for 24 h without the addition of isopropyl 1-thio--D-galactopyranoside. Following induction, cells were harvested by centrifugation at 3000 x g for 20 min and stored at –80 °C pending purification.
RavA was purified using Qiagen nickel-nitrilotriacetic acid-agarose beads according to the manufacturer's protocols. The HV tag was removed by digestion with tobacco etch virus protease at 1:10 molar ratio of tobacco etch virus to protein for 5 h at 4°C. The protein was further purified using a Mono S HR 5/5 column.
ViaA was precipitated from cell lysate using 30% ammonium sulfate. Pellets were resuspended and exchanged into a no-salt buffer using PD10 desalting columns. The protein was further purified on a Heparin HP5 column.
Cells overexpressing LdcI were lysed in buffer LI1 (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1 mM pyridoxal 5'-phosphate, 5% glycerol, and 1 mM DTT). Cell debris was removed by centrifugation. The supernatant was heated at 70 °C for 5 min resulting in the precipitation of the bulk of E. coli proteins, however, most of LdcI remained soluble. Precipitate was removed by centrifugation at 30,000 x g for 15 min. LdcI in the supernatant was further purified by ion exchange chromatography using a Mono Q HR 5/5 column and then by size exclusion chromatography using a Superdex 200 HR 10/30 column.
LdcC was precipitated from cell lysate using 30% ammonium sulfate, and the pellets were resuspended and exchanged into low salt buffer by dialysis. The protein was further purified using a Mono Q HR 5/5 column.
The final yields of purified proteins per 1 liter of culture were as follows: 6 mg for RavA, 2 mg for ViaA, 10 mg for LdcI, and 5 mg for LdcC. Proteins were judged to be >95% pure by SDS-PAGE analysis. All chromatography columns used were from Amersham Biosciences. Protein concentration was determined using the Bio-Rad Protein Assay.
Measurement of RavA ATPase Activity—RavA ATPase activity was measured by determining the free phosphate released after ATP hydrolysis using a colorimetric assay similar to that reported previously (35). Briefly, RavA was placed in buffer RA1 (5 mM MgCl2, 0.02% Triton X-100, and 1 mM DTT) containing various concentrations of ATP substrate. RA1 solutions of different pH were generated using 10 mM concentrations of citrate, acetate, MES, HEPES, or TAPS buffers. Where applicable, BSA, ViaA, LdcI, and LdcC were included in the buffer. Assays were performed in 100-µl volumes at 37 °C in Eppendorf tubes or in a quartz plate. At different time points, 10-µl aliquots of the assay mixture were removed and added to 200 µl of RA2 (1 mM malachite green, 8.5 mM ammonium molybdate, and 1 M HCl). Color development was allowed to proceed for 1 min and then was stopped by the addition of 25 µl of 37% citric acid. Absorbance was measured at 660 nm and converted to moles of phosphate produced using a KH2PO4 standard curve.
Preparation of Cell Extracts for LdcI Activity Assays—WT and ravA cells were grown in LB media containing 0.4% glucose to an A600 of 3.0–4.0 at 37 °C. Cells were pelleted by centrifugation, resuspended in lysis buffer LT1 (25 mM HEPES, pH 7.0, 300 mM NaCl, 10% glycerol, and 5 mM -mercaptoethanol), and lysed by sonication. The lysates were cleared of debris by centrifugation, flash frozen on liquid N2, and stored at –80 °C until use in assays.
Measurement of LdcI Lysine Decarboxylase Activity—The ability of LdcI to generate cadaverine from lysine was measured as previously described with slight modification (36). Purified LdcI was dissolved in buffer LA1 (2.5 mM MgCl2, 1 mM ATP, 0.1 mM PLP, and 4 mM lysine). LA1 solutions of different pH levels were generated using acetate, HEPES, or TAPS buffers at a concentration of 50–100 mM. Total cell protein (25 µg/assay) was dissolved in buffer LA2 (25 mM MES, pH 6.0, 0.1 mM PLP, and 4 mM lysine). Assays were conducted in 100-µl volumes at 37 °C using either a quartz plate or Eppendorf tubes. At specific time points, 15-µl aliquots were removed and added to an equal volume of 1 M Na2CO3 stop solution. Subsequently, 15 µl of 10 mM 2,4,6-trinitrobenzenesulfonic acid (TNBS) was added, and samples were heated at 40 °C for 5 min to allow the formation of cadaverine-TNBS and lysine-TNBS adducts. Cadaverine-TNBS (N,N'-bistrinitrophenylcadaverine) was extracted using 500 µl of toluene, and the absorbance at 340 nm was measured. An extinction coefficient of 2.5 x 104 M–1 cm–1 was used (36) to convert absorbance into moles of cadaverine produced.
Growth in LdcI Indicator Media—E. coli K12 MG1655 WT, ravA, and cadB cells were grown in lysine decarboxylase indicator media DI1 (0.3% yeast extract, 0.1% glucose, 0.5% lysine, and 0.0016% bromocresol purple pH indicator) at 37 °C. Samples were taken at selected time points, cells were pelleted by centrifugation, and the absorbance at 590 nm of the bromocresol purple indicator in the cleared lysates was measured.
Extreme Acid Shock Assays—Extreme acid shock assays were adapted from previously described protocols (37). Briefly, E. coli K12 MG1655 WT and ravA cells were grown in LB media containing 2% glucose at 37 °C for 18 h. Cells were diluted 1/100 into pH 2.5 acid shock media AS1 (40 mM KCl, 80 mM KH2PO4, 33 mM H3PO4, 1.7 mM sodium citrate, and 20 mM glucose) with and without 1 mM lysine. Samples were incubated at 37 °C for 2 h and then serially diluted in LB media in a 96-well plate. Diluted cells were then spotted onto an LB agar plate and grown up at 37 °C overnight. Growth on plates was examined to determine cell viability.
Northern Blot Analysis—E. coli MG1655 cultures were grown in LB media at 37 °C to an A600 of 0.6–0.8. Total RNA was isolated using the single step acid guanidinium thiocyanate-phenol-chloroform extraction method as described (38). Briefly, 1.5-ml samples of culture were centrifuged and resuspended in 500 µl of buffer NB1 (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sodium lauryl sarcosinate, and 0.1 M -mercaptoethanol). Samples were then passed four times through a 25-gauge needle to facilitate lysis. 0.05 ml of 2 M sodium acetate (pH 4.0), 0.5 ml phenol, and 0.1 ml chloroform-isoamyl alcohol were then added sequentially to the sample. After each addition, the tube was capped and mixed thoroughly by inversion. Samples were then vortexed briefly, incubated on ice for 15 min, and centrifuged at 10,000 x g for 20 min at 4 °C. The upper aqueous phase was transferred to a fresh tube, and an equal volume of isopropanol was added. The solution was mixed thoroughly and incubated at –20 °C for 1 h. RNA precipitate was collected by centrifugation at 10,000 x g for 30 min at 4 °C. The isopropanol was carefully decanted, and the pellet was resuspended in 0.15 ml of buffer NB1. After thorough mixing, the sample was precipitated again using isopropanol. After centrifugation, the RNA pellet was washed twice with 75% ethanol, and the pellet was resuspended in 50 µl of diethyl pyrocarbonate water. Samples were stored at –80 °C.
20 µg of total RNA was separated on a 1.2% agarose gel containing 6.7% formaldehyde. RNA was transferred to Hybond N+ Nylon membrane (Amersham Biosciences) and was covalently cross-linked to the membrane by UV irradiation using a Stratalinker UV Crosslinker (Stratagene). Probe generation and Northern blotting were carried out using the AlkPhos Direct Labeling and Detection System (Amersham Biosciences). For RavA, the DNA probe used was complementary to bp 623–1497 of the ravA gene, whereas for ViaA, the DNA probe used was complementary to bp 719–1452 of the viaA gene.
RT-PCR—RT-PCR analysis was performed using the Qiagen OneStep RT-PCR kit as described in the manufacturer's instructions. 2 µgof E. coli K12 MG1655 total RNA was used as a template. Amplification was carried out using primers RAVA_forward2 (5'-tatcacggcgccatggctcaccctcatttattag-3') and VIAA_reverse and primers RAVA_internal_forward (5'-gcgaattccatatgcaacaaattgatgtattgatgaccg-3') and VIAA_internal_reverse (5'-cgttcgatcactttttcacg-3').
Western Blot Analysis—Typically, cell pellets were lysed by sonication, and lysates were centrifuged to remove cellular debris. Supernatant was transferred to a fresh tube, and protein concentration was determined using the Bio-Rad Protein Assay. 5–40 µg of proteins was separated on a 10% SDS-PAGE gel and transferred to Biotrace polyvinylidene difluoride membrane using a Bio-Rad semidry transfer apparatus as per the manufacturer's instructions. Membranes were then incubated with rabbit polyclonal anti-RavA or anti-LdcI antibodies generated at the Division of Comparative Medicine, University of Toronto. Membranes were then washed and incubated with protein LA-peroxidase. ECL detection reagent (Amersham Biosciences) was used to visualize RNA bands on a Bioflex chemiluminescence detection film (Clonex).
Subcellular Fractionation of E. coli—200 ml of cells grown at 37 °C until an A600 of 2.5 was divided into 2 x 100 ml and centrifuged at 4000 x g for 10 min at 4 °C. Cell pellets were washed once with buffer SF1 (10 mM Tris-HCl, pH 7.1, and 30 mM NaCl) and then centrifuged again. One pellet was used for the isolation of perisplasmic and cytoplasmic proteins, and the other pellet was used for the isolation of membrane proteins. Periplasmic and cytoplasmic proteins were isolated using a protocol based on a previously described osmotic shock method (39), whereas total membrane proteins were isolated as previously described (40).
Size Exclusion Chromatography—A calibrated Superose 6 HR 10/30 column connected to an AKTA fast-protein liquid chromatography system (Amersham Biosciences) was used for size exclusion chromatography. Samples were typically run in buffer SE1 (25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 2.5 mM MgCl2, and 1 mM DTT) in the presence or absence of 1 mM ATP. 0.1 mM PLP was added to the buffer when LdcI or LdcC were included in the sample. The column was calibrated using molecular mass standards (Sigma): thyroglobulin (669 kDa), apoferritin (443 kDa), -amylase (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). All experiments were performed at 10 °C, and absorbance was monitored at 280 nm.
Analytical Ultracentrifugation Analysis of RavA—Sedimentation equilibrium experiments on RavA were carried out at the Ultracentrifugation Service Facility in the Department of Biochemistry at the University of Toronto. 20 µM RavA protein in Buffer AU1 (25 mM Tris, pH 7.5, 300 mM NaCl, 1 mM DTT, and 10% glycerol) containing 0.2 mM ATP was spun at 5000 and 8000 rpm, at 4 °C, in an An-60 Ti rotor in a Beckman Optima XL-A analytical ultracentrifuge. Absorbance was recorded at 230 and 280 nm. Data analysis was performed using the Origin MicroCal XL-A/CL-I Data Analysis Software Package Version 4.0.
Mapping of RavA Domains—7.5 µg of purified RavA protein was dissolved in 140 µl of buffer RD1 (10 mM Tris, pH 7.5, 100 mM KCl, 10 mM MgCl2, 2.5 mM DTT, and 5 mM ATP) and digested with 0.075 µgof trypsin (Roche Applied Science, 1418475) at 4 °C. At specific time points aliquots of digested protein were removed and separated using SDS-PAGE. Sample preparation of bands for mass spectrometry was adapted from a previously described method (41). Briefly, the samples were processed in 500 µl of PCR grade tubes. The gel band was cubed and washed sequentially in high-performance liquid chromatography grade water (VWR, EM-WX0004-1) and 100 mM ammonium bicarbonate (Sigma, A6141). The gel cubes were dehydrated between the following steps with 100% acetonitrile (VWR, EM-AX0151-1): gel cubes were reduced with 1 mM DTT (Sigma D9163) at 50 °C for 30 min and alkylated with 55 mM iodoacetamide (Sigma, I1149) at room temperature for 20 min in the dark. Gel cubes were dehydrated with acetonitrile washes and SpeedVac-treated to remove acetonitrile. Protein was digested by swelling the cubes with 50 µl of 0.1 ng/µl trypsin in 50 mM ammonium carbonate and 0.1% calcium chloride overnight. The tryptic peptides were extracted from the gel slices by shaking with two washes of 150 µl of 100 mM ammonium carbonate buffer at 37 °C for 45 min. Liquid from the overnight digestion and two extractions were pooled on ice and acidified with 2 µl of 1% glacial acetic acid (VWR EM-AX0073-6). Peptides were desalted and concentrated using C18 resin (Sigma, H8261) by a batch method. Final volume was 10 µl of extracted tryptic peptides in 65% acetonitrile and 1% acetic acid. A 1-µl aliquot of tryptic peptide sample was mixed with 1 µl of a saturated 
-cyano-4-hydroxycinnamic acid (Sigma, C2020) solution in 30% acetonitrile and 1% acetic acid and spotted onto a stainless steel matrix-assisted laser desorption ionization sample plate (Bruker Daltonics). For high mass bands, the samples were concentrated and desalted by a batch method similar to tryptic peptide clean up but with the exception that C4 resin (Sigma) was used. The final volume was 15 µl in 65% acetonitrile and 1% acetic acid. A 1-µl aliquot of partial proteolysis sample was mixed with 1 µl of a saturated sinapinic acid (Sigma, D7927) solution in 30% acetonitrile and 1% acetic acid and spotted onto a stainless steel matrix-assisted laser desorption ionization sample plate.
High mass samples were analyzed in a Bruker Reflex III (Bruker Daltonics) in high mass linear mode with a high mass detector. Carbonic anhydrase (Sigma, C3934) and lysozyme (Sigma, L7651) were used as external calibrants. Tryptic peptides were analyzed in a Bruker Reflex III (Bruker Daltonics) in reflector mode. Samples were internally calibrated using trypsin peptide peaks. Mass mapping was done using PAWS (Genomic Solutions) against the known sequence of the protein. The tryptic peptide fingerprint of a gel band was used to determine an approximate region for the partial proteolysis fragment from the full sequence. The observed high mass was then used to define the exact cut sites for the partial proteolysis fragments.
Electron Microscopy—For preparation of negatively stained protein samples, 3 µl of protein solutions was applied to glow-discharged continuous carbon copper grids and left for 1 min before washing with two drops of 1% (w/v) uranyl acetate. The grids were observed with a Philips CM 12 transmission electron microscope with an LaB6 filament at 80 kV. Images were recorded on Kodak SO-163 films at 35,000x magnification in the case of RavA, LdcI, and ATP
S·RavA·LdcI preparations and at 22,000x for the ADP·RavA·LdcI preparation. Negatives were digitized on a Zeiss SCAI scanner at a pixel size of 7 µm, corresponding, at the specimen level, to 0.2 nm and 0.318 nm at 35,000x and at 22,000x, respectively. Image processing was carried out on a Linux workstation using the EM (42, 43), EMAN (44), BSOFT (45), and PFT2 (46)5 software packages. Images were binned to 0.4 nm or to 0.636 nm at the specimen level.
Image Analysis—For LdcI, 3631 subframes of 88 x 88 pixels containing single particles were extracted interactively from several micrographs at similar defocus of 
600 nm, and low-pass-filtered at 17 Å resolution to remain within the first zero of the contrast transfer function. This data set was translationally but not rotationally aligned relative to the rotationally averaged total sum of the individual images. The aligned data set was subjected to multivariate statistical analysis/EM, which clearly demonstrated the 5-fold symmetry of the LdcI particle. Characteristic class averages were then used as a set of references for multireference alignment/EM followed by multivariate statistical analysis and classification. The resulting class averages were similar to those obtained by reference free alignment and classification routine in EMAN. Five characteristic views were selected to generate an initial three-dimensional model of the LdcI particle by cross common lines. Refinement of the three-dimensional model was, therefore, undertaken with a D5-symmetry imposed (EMAN and PFT2/BSOFT). After convergence, the symmetry was relaxed and the absence of divergence was verified. The resolution of the reconstruction was determined via Fourier shell correlation to be 20 Å according to the 0.5 criterion. The handedness was not determined.
For ADP·RavA·LdcI and ATPS·RavA·LdcI complexes, the procedure was similar to the one described above for LdcI alone. 2902 subframes of 80 x 80 pixels (6.36 Å/pixel) were extracted from the images of ADP·RavA·LdcI and 2629 subframes of 112 x 112 pixels (4 Å/pixel) from the images of ATPS·RavA·LdcI. The data were low-pass-filtered at 18 Å, which is again within the first zero of the contrast transfer function. In this case, the majority of particles were present in a side-view orientation or as tilted views. Given the appearance of the side views it was obvious that the RavA·LdcI complex looked like a sandwich of two LdcI molecules bridged together by densities that might be attributed to RavA. Therefore, the reconstruction was performed imposing a 5-fold symmetry of the complex, although, contrary to the situation with LdcI alone and due to the under representation of top views, it could not be revealed by multivariate statistical analysis. Once again, classification allowed selection of five characteristic views used for generation of an initial 5-fold symmetric model by cross common lines. In addition, two alternative three-dimensional models were created by using the same five characteristic views supplemented by a simulated top view. The two simulated top views were composed of the previously determined top view of LdcI alone surrounded by five symmetrically distributed blobs of density positioned either as a radial extension of each LdcI subunit or in the middle between two adjacent subunits to reach the dimensions compatible with the observed side views of the RavA·LdcI complexes. After the refinement in EMAN and PFT2/BSOFT with a D5-symmetry imposed, all the models converged to the same final structure, which remained stable upon symmetry relaxation. The reconstructions of the two complexes ADP·RavA·LdcI and ATPS·RavA·LdcI looked indistinguishable from each other and had a similar final resolution. The resolution of the reconstructions was estimated to be 30 Å via Fourier shell correlation but was most probably asymmetric due to the preferential adsorption of the particles on the carbon surface in a side-view orientation. Moreover, if some of the nucleotide·RavA·LdcI complexes were built by LdcI molecules linked not by five but by four or three RavAs, for example, such heterogeneity of the preparation would also lead to a deterioration of the achievable resolution.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Examination of a ClustalW (27) alignment of all 36 RavA sequences revealed the presence of a number of highly conserved signature sequences in the E. coli K12 MG1655 RavA sequence (marked in red in Fig. 2). Upon comparison to common AAA+ signature sequences (1), it is observed that many of these conserved residues distinguish RavA proteins from other AAA+ proteins. At the N terminus, the Box II motif contains two highly conserved residues, an Arg and hydrophobic residue (Leu in the RavA sequence). The Walker A motif contains a number of highly conserved residues, but most of these are common to all AAA+ proteins. One feature of note, however, is that members of the RavA subfamily favor an alanine residue in place of the more commonly observed glycine of the classical "GK(S/T)" motif. A series of highly conserved residues also exist in the Box IV and Box IV' motifs. Based upon comparison to published alignments, at least part of the sequence in this region may lie on a small "insert" in the second helix of the AAA+ core module (2). The Walker B motif also contains a number of highly conserved residues. Most of these are common to all AAA+ proteins, however, the conserved tryptophan/phenylalanine residue is particularly unique to RavA and may have important functional significance to RavA proteins. A unique, highly conserved LP motif also exists immediately prior to the Walker B motif.
Within Box VI, which follows the Walker B, members of the MoxR family possess a highly conserved LLXX
XE motif, with members of the RavA subfamily possessing a slightly extended version IXNXLLXXNE(R/K) motif, where corresponds to Ile, Leu, Val, or Met. The significance of this conserved region is unclear. The Sensor 1 region contains a highly conserved PX5ASNXP motif. The conserved Asn residue appears to correspond to the AAA+ Sensor 1 residue (5), responsible for interacting with the gamma phosphate of bound ATP. The conserved Ser is also very common in AAA+ in general. The rest of this conserved sequence, however, appears to be specific to RavA/MoxR proteins.
A conserved LXAXYDRXXXR motif exists in Box VII-Box VII' region. This region contains the conserved arginine finger of AAA+ proteins, believed to be responsible for mediating intersubunit communication in response to ATP hydrolysis (5). The first Arg residue in the conserved motif may correspond to the arginine finger. Some additional conserved hydrophobic residues also exist in Box VII' and in the sequence following Box VII''. The Sensor 2 region contains a short stretch of conserved residues, SDRR. One of the arginines is expected to correspond to the conserved Sensor 2 residue present throughout most of the AAA+ superfamily. This residue is believed to be responsible for sensing ATP and mediating movements between the AAA+ core domain and small -helical domain (5). The Ser residue and first Arg are common to many MoxR proteins in general, however, the Asp and second Arg residues appear to be unique to RavA subfamily members and may be important for a specific function in these proteins. Some additional conserved hydrophobic residues also exist in this region, however, there is little conservation in the RavA sequences after Sensor 2.
RavA Is a Soluble Cytoplasmic ATPase Consisting of Two Domains— To experimentally determine the domains present in RavA structure, we expressed and purified the protein from E. coli. The purified protein was then treated with limited amounts of sequencing grade trypsin for varying amounts of time (Fig. 3A). The boundaries of the fragments generated were then determined using mass spectrometry (Fig. 3B). Based on this analysis, the protein can be divided into two main domains: a highly conserved N-terminal domain (Met1–Arg347), which corresponds to the AAA+ module, and a poorly conserved C-terminal domain (Arg348–Cys498) that is predicted to be predominantly -helical. As is typical for AAA+ modules (1), the N-terminal domain can be separated into two smaller fragments (Fig. 3, A and B), which appear to correspond to the AAA+ subdomain (Met1–Arg187) and an -helical subdomain (Ser188–Arg347). The C-terminal domain of RavA has no known motifs or signature sequences and appears to represent a novel domain of unknown function. The sequence of this domain bears little homology to any other protein sequence in NCBI (48) and is poorly conserved in RavA proteins outside of the enterobacteria subdivision. It is interesting to note that the E. coli RavA sequence after residue Ala288 is highly enriched in glutamines (Fig. 2).
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ravA cells grown at 37 °C to early stationary phase were lysed and separated into cytoplasmic, periplasmic, and crude membrane fractions. Western blot analysis of these fractions using RavA antibodies (Fig. 3C) clearly showed that RavA is predominantly cytoplasmic. This is consistent with the lack of any predicted signal sequences or transmembrane regions in RavA. A faint band observed in the membrane fractions appears to be nonspecific, as it appears in both the WT and ravA strains. As shown in Fig. 3D, purified RavA at 0.4 µM has the highest ATPase activity at neutral to alkaline pH, with the highest activity around pH 7.5. Below pH 7.5, the activity appears to drop rapidly and is minimal below pH 6.0. Complete Michaelis-Menten kinetic analysis was performed at pH 7.5 (Fig. 3D, inset). The kcat and Km values for RavA ATPase are in a similar range to those obtained for other AAA+ proteins such as HslU (49), NtrC (50), and Lon (51). Furthermore, RavA also has GTPase activity. GTP hydrolysis appears to be much slower than ATP hydrolysis at lower enzyme/substrate concentrations but reaches comparable levels with increasing amounts of enzyme/substrate (data not shown). This effect may be the result of a lower ability of GTP versus ATP to promote RavA oligomerization (data not shown).
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S— The ravA and viaA gene sequences encode predicted proteins of similar length consisting of 498 and 483 amino acid residues, respectively. The genes reside on the E. coli chromosome close to the origin of replication (OriC) (52), 2200 bp clockwise from OriC, and the genes are transcribed in the counterclockwise direction. Interestingly, the 5'-end of the ViaA coding region overlaps the 3'-end of the RavA coding sequence by seven bases (Fig. 4A). Such an overlap might hint to the presence of an ancient gene whose sequence was a fusion of the ravA and viaA gene sequences.
To determine whether ravA and viaA are on the same operon, Northern blot analysis was carried out on E. coli MG1655 wild-type (WT), MG1655 ravA, and MG1655 viaA cells. The ravA cells have most of the RavA ORF deleted: from 13 bp upstream of the `atg' start codon to bp 1431 (Fig. 4B). This deletion does not disrupt the ViaA ORF. The viaA cells contain a deletion of bp 166–1437 of the ViaA ORF. The cells were grown in LB media at 37 °C to mid-log phase; subsequently, total RNA was isolated and probed with alkaline phosphatase-labeled DNA probe complementary to bp 623–1497 of the ravA gene or complementary to bp 719–1452 of the viaA gene (Fig. 4, A and B). Wild-type cells clearly show the presence of two different ravA mRNA transcripts of 1900 and 1100 bases in size (Fig. 4C, bands 2 and 3, respectively, in the first lane of panel I). Low levels of an even larger transcript of 3100 bases are also detected (Fig. 4C, band 1 in the first lane of panel I). RT-PCR analysis using forward and reverse primers complementary to the 5'-end of the ravA ORF and the 3'-end of the viaA ORF, respectively, confirms the existence of the larger transcript (Fig. 4D, band 1). In addition, RT-PCR using a forward primer internal to the ravA ORF and a reverse primer internal to the viaA ORF also produces a product (Fig. 4D, band 2), further supporting the existence of these two genes on a single transcript. viaA transcript is detected as distinct bands of 2000 and 1200 bases (Fig. 4C, bands 1 and 2, respectively, in first lane of panel II). A larger transcript is not detected in the viaA blot, likely a reflection of lower probe sensitivity.
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3100 bases transcript appears to be inherently unstable, however, and undergoes at least two distinct internal cleavage events shown schematically in Fig. 4E. The first of these cleavages produces the 1900-base fragment observed in the ravA blot (Fig. 4C, band 2 in the first lane of panel I) and the 1200-base fragment observed in the viaA blot (Fig. 4C, band 2 in the first lane of panel II). The second cleavage produces the 1100-base fragment observed in the ravA blot (Fig. 4C, band 3 in the first lane of panel I) and the 2000-base fragment observed in the viaA blot (Fig. 4C, band 1 in the first lane of panel II). Whether or not these cleavage events have a regulatory role is currently under investigation.
Northern blot analysis of MG1655 ravA show that the deletion of the ravA gene results in the loss of all mRNA species (Fig. 4C, lane 2 in both panels). On the other hand, Northern blot analysis of MG1655 viaA using the probe for ravA shows the presence of an 1700-base transcript and the previously observed 1100-base transcript (Fig. 4C, bands 4 and 5, respectively, in lane 3 of panel I). The 1700-base transcript is slightly smaller than the 1900-base fragment observed in wild-type cells (Fig. 4C, band 2 in lane 1 of panel I) probably resulting from earlier termination of transcription due to the presence of the deletion cassette (Fig. 4B). These results confirm that the ravA and viaA genes form a single operon.
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S promoter consensus sequence (Fig. 4A) (53). This consensus sequence has been shown to be present in genes that are up-regulated in stationary phase. Consistent with this, Western blot analysis of MG1655 cells shows a significant induction of RavA levels as cells approach late log/early stationary phase (Fig. 4F, left panel). Such an induction does not occur in an MG1655 rpoS strain, although constant levels of RavA protein appear to be present at all times (Fig. 4F, right panel). The data suggest that RavA induction is partially dependent on the stationary phase sigma factor S, likely in a direct manner. The presence of low, constitutive levels of RavA protein in the rpoS strain suggests that the ravA promoter may display conditional selectivity allowing its expression to be regulated by other sigma factor(s) (e.g. 70). Such conditional promoter selectivity has been reported for other E. coli genes, for instance dps and yaiA, whose expression may be regulated by either S or 70, depending on cellular conditions (53).
RavA Forms a Hexameric Oligomer—Many AAA+ proteins typically function as oligomeric rings (6); therefore, the nature of the oligomeric state of RavA was investigated using size exclusion chromatography and analytical ultracentrifugation. Size exclusion chromatography analysis using 3 µM RavA revealed that the protein forms oligomeric species and that this oligomerization is enhanced in the presence of nucleotide (Fig. 5A). The largest species formed runs near the 443-kDa marker. To more accurately confirm the size of this species, analytical ultracentrifugation sedimentation equilibrium studies were performed in the presence of ATP. The best fit to the data set corresponds to a monodisperse species of 310 kDa in size (Fig. 5B). This molecular weight corresponds closely with that expected of a hexameric species. Thus we propose that the functional state of RavA is a hexameric oligomer in the presence of nucleotides. Sedimentation equilibrium experiments in the absence of nucleotides revealed the existence of a range of smaller species (data not shown), consistent with our size exclusion results.
Negative stain electron microscopy of RavA in the presence of ADP, ATP, or non-hydrolyzable ATP analogues invariably showed a mixture of different oligomeric states of the protein, ranging from monomers to bigger oligomers. The top-view appearance of the largest oligomers clearly favored their hexameric composition (Fig. 5C). However, due to the inhomogeneity of RavA oligomeric states on the grid and the resulting difficulty in unambiguous identification of corresponding side views, no attempts have been made to obtain a three-dimensional reconstruction of the RavA hexamer (however, see below).
RavA Binds to the Inducible Lysine Decarboxylase LdcI/CadA—In an effort to determine the function of RavA, experiments were carried out in an attempt to identify its interacting partners. E. coli DY330 strain (30, 31), in which a TAP tag (54) was fused C-terminally to the endogenous ravA chromosomal gene, was used for pull-down experiments (33). When RavA-TAP was isolated from cells grown to stationary phase in TB-rich media at 37 °C, the inducible cytoplasmic lysine decarboxylase (LdcI) was found to be strongly bound to RavA (Fig. 6A). LdcI (also termed CadA, SwissProt name DCLY_ECOLI, SwissProt number P0A9H3) is a pyridoxal phosphate (PLP, a vitamin B6 derivative)-dependent decameric enzyme (55, 56), which plays a major role in the acid stress response in bacteria (57–59). LdcI decarboxylates a lysine to produce the polyamine cadaverine, which is then pumped out of the cell by an inner membrane lysine/cadaverine antiporter protein, CadB (59, 60). This process removes a proton from the cell cytoplasm and reduces the acidification of the intracellular environment. The genes cadB and ldcI/cadA are on the same operon (60, 61), and the operon is activated by an inner membrane anchored transcription activator, CadC, that binds a Pcad element in the promoter region of cadBA (61). The regulation of cadBA transcription is rather complex. The operon is induced by low external pH, by the presence of exogenous lysine, and under anaerobic conditions (55, 62). The binding of RavA to LdcI leads us to speculate that RavA might also be involved in the bacterial stress response or that the interaction between the two proteins is regulatory in nature.
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440 kDa representing the hexameric species (Fig. 6B, panel 1, top panel). 10 µM LdcI (Fig. 6B, panel 2) also elutes as a single species, with an apparent molecular mass of 600 kDa probably representing the decameric species that is known to be formed by LdcI (also see below) (55, 56). A mixture of 10 µM LdcI with excess RavA (18 µM) results in a large shift in the migration of both species indicating a strong interaction (Fig. 6B, panel 3). A range of higher order oligomers are formed, with the predominant complex (boxed) migrating at or near the void volume of the column, suggesting a size of 50 MDa or greater. The predominant complex formed changes if 18 µM RavA is incubated with 2-fold excess of LdcI (40 µM). The formed complex elutes within the column volume, and its molecular mass is 3–5 MDa as estimated by sedimentation equilibrium experiments (Fig. 6B, left box in panel 4 and data not shown). We term this complex the fraction three complex (FTC), which, as discussed below, might be the physiologically relevant RavA·LdcI complex. E. coli contains another protein that is a very close paralogue of LdcI. This protein is the constitutive lysine decarboxylase LdcC (SwissProt name DCLZ_ECOLI, SwissProt number P52095 [GenBank] ). LdcI and LdcC are 69% identical and 84% similar as determined using pairwise BLAST P analysis. LdcC is expressed in E. coli at very low levels, and its exact role in the cell remains unclear (63, 64). Like LdcI, LdcC is also a PLP-dependent enzyme that decarboxylates lysine and possibly assembles into a decamer (63, 64) (Fig. 6B, panel 5). However, the enzymatic activity profile and stability of LdcC differ from that of LdcI (63, 64). Due to the high sequence similarity between LdcC and LdcI, we expected that LdcC might also bind RavA, however, we were surprised to find that there is no significant interaction between RavA and LdcC (Fig. 6B, panel 6). The specific interaction of RavA with LdcI and not LdcC possibly indicates that RavA is a stress response protein.
The size of the LdcI oligomer (decamer) is unaffected by the presence or absence of ATP (Fig. 6C, panel 1, upper panel) or by LdcI concentration (compare elution profile of 10 µM LdcI in Fig. 6B, panel 2, to the elution profile of 1.5 µM LdcI in Fig. 6C, panel 1). As shown in Fig. 6C, the size of the RavA·LdcI complex is dependent upon RavA oligomerization. 3 µM RavA in the absence of ATP predominantly elutes at 60 kDa representing a monomeric species, whereas, in the presence of ATP, 3 µM RavA elutes as the larger hexameric species (Figs. 5A and 6C, panels 2 and 3). LdcI binds to 3 µM RavA in the absence and presence of ATP (Fig. 6C, panels 4 and 5), however, the size of the complex is smaller in the absence of ATP as compared with when ATP is present. These data indicate that LdcI probably does not promote the oligomerization of RavA in the absence of nucleotide. In Fig. 6C (panel 5), the FTC is also observed.
Similar interactions were observed between RavA and LdcI when ADP was added to the buffer rather than ATP. Furthermore, the inclusion of lysine or cadaverine or the removal of the LdcI cofactor PLP from the buffer did not affect the interaction between RavA and LdcI (data not shown). These observations clearly indicate that the interaction between RavA and LdcI is highly robust and functionally significant.
RavA·LdcI Forms a Very Large Distinctive Cage-like Complex as Visualized by Negative Stain Electron Microscopy—A 20 Å resolution structure of the D5-symmetric LdcI oligomer (Fig. 7, A–C) was obtained as described under "Materials and Methods." The existence of a 5-fold symmetry axis in the particle was corroborated by multivariate statistical analysis. Fig. 7B shows the pronounced 5-fold symmetrical shape of the top-view class average and the 2-fold symmetry of the side-view class average. Some dimeric dissociation products could always be detected in the background of the micrographs (Fig. 7A), in agreement with the early publication of Sabo et al. (55). The random orientation of single particles on the carbon film made this specimen particularly suitable for a three-dimensional reconstruction by angular reconstitution approach (Fig. 7C). The decamer of LdcI revealed itself as two pentameric rings, tightly stacked together back-to-back and turned 35° with respect to each other. Each monomer has a globular shape. The relative rotation of the two rings confers a pronounced handedness to the molecule responsible for the "plait-like" appearance of the side view.
Grids prepared by mixing ADP·RavA or ATPS·RavA and LdcI in equimolar amounts (1.8 µM RavA to 1.8 µM LdcI) looked indistinguishable from preparations of LdcI alone except for a higher protein background attributed to RavA. Remarkably, however, at 2-fold excess of RavA (3.24 µM RavA and 1.8 µM LdcI in the presence of 3 mM ADP or ATPS), the background became clean, individual LdcI views virtually disappeared, and most of the protein was present as an amazingly large and apparently homogeneous RavA·LdcI complex. Analysis of the data revealed that the ADP·RavA·LdcI complex contained not only one, but two LdcI decamers sandwiched together and glued by additional densities that certainly arise from RavA (Fig. 7D). In this case, however, the particles had a strongly preferred orientation on the grid, and although different class averages could be easily identified as side views and various tilted views, none could be attributed to a true 5-fold symmetrical top view. Nevertheless, even without a top view class average, an initial model could still be constructed from several characteristic tilted views (Fig. 7E) by cross common lines technique, provided a 5-fold symmetry is imposed. A refinement with such a model resulted in the reconstruction shown in Fig. 7F. The validity of this approach was confirmed by creating two of the possible top views of the complex, composed of the experimentally observed top view of the LdcI alone that was artificially surrounded by five blobs of density. This density was arbitrarily positioned either in the middle between two adjacent LdcI subunits or as a radial extension of each LdcI subunit (see the first two panels in Fig. 7E). The only restrictions were the 5-fold symmetry of the top view and the experimental dimensions of the ADP·RavA·LdcI complex side view. These two additional sets of views gave rise to two different initial three-dimensional models of the complex, which converged during the refinement process to the same final reconstruction (Fig. 7F), identical to the one obtained without any artificial top view.
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S (data not shown), at least at the current resolution estimated to be 30 Å. We believe that the complex seen in Fig. 7 corresponds to the FTC in Fig. 6.
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Furthermore, LdcI activity in whole cell extracts was also not affected by deletion of the ravA gene (Fig. 8B), in which the enzyme was induced by growth in unbuffered LB media containing excess glucose. The utilization of glucose as a carbon source results in the decrease in the pH of the media as cells ferment the glucose and produce lactic and acetic acids (65). The addition of purified RavA and nucleotides to the extract also did not affect LdcI activity (data not shown). The lack of effect of RavA on LdcI activity in vivo was further confirmed by comparing the ability of WT and ravA cells to recover from a decrease in pH using the CadBA system by growing cells in special indicator media (Fig. 8C). This media contains excess glucose, bromocresol purple as a pH indicator, and lysine for use by the CadBA system. The change in the pH of the medium over time can be tracked by monitoring the change in the absorbance of the indicator at 590 nm. As shown in Fig. 8C, both WT and ravA cells experience an initial decrease in pH from 6.1 to 5.0 but then recover, eventually raising the pH of the medium back to pH 6.1. On the other hand, a cadB mutant, which is also deficient in LdcI/CadA production (data not shown), cannot restore the pH of the media, showing that pH recovery is dependent upon a functional CadBA system. Both WT and ravA strains have equal recovery rates, showing that the lysine-dependent response to low pH does not appear to be compromised in the knock-out strain.
The ability of WT and ravA cells to respond to a more extreme acid challenge was also examined. Cells were grown to stationary phase in LB media and then exposed to minimal media at pH 2.5, with and without added lysine, for 2 h. Cells were then diluted in LB media and plated on LB agar plates to measure cell viability (Fig. 8D). Both WT and ravA cells appear to withstand this extreme acid stress equally well in the presence of lysine, providing further evidence that the CadBA system does not appear to be compromised in the ravA deletion strain. Finally, although LdcI is strongly induced when cells are shifted from neutral pH to pH 5.5, no such induction is observed for RavA (Fig. 8E). RavA is also not induced by shift to high pH media (data not shown). The induction of LdcI or its levels were also not affected in ravA deletion or overexpression strains (data not shown). All the data presented above seem to strongly suggest that the RavA·LdcI complex does not function to regulate LdcI activity, but, rather,
