Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase
2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês
10.1074/jbc.m708620200
ISSN1083-351X
AutoresJonathan A. R. Worrall, Franklyn A. Howe, Adam R. McKay, Carol V. Robinson, Ben F. Luisi,
Tópico(s)RNA Research and Splicing
ResumoHelicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696–762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpy-favored interaction between the helicase and RNase E-(696–762) with an equilibrium binding constant (Ka) of at least 1 × 108 m-1. We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function. Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696–762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpy-favored interaction between the helicase and RNase E-(696–762) with an equilibrium binding constant (Ka) of at least 1 × 108 m-1. We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function. RNA helicases are a diverse set of proteins found in all three kingdoms of life that possess the ability to unwind short stretches of RNA duplexes in reactions that require the hydrolysis of nucleoside triphosphates (1Cordin O. Banroques J. Tanner N.K. Linder P. Gene (Amst.). 2006; 367: 17-37Crossref PubMed Scopus (702) Google Scholar). They are the largest group of enzymes in eukaryotic RNA metabolism and are involved in virtually all aspects of cellular RNA manipulation including transcription, splicing, RNA nuclear export, and ribosome biogenesis (2Anantharaman V. Koonin E.V. Aravind L. Nucleic Acids Res. 2002; 30: 1427-1464Crossref PubMed Scopus (382) Google Scholar, 3Bleichert F. Baserga S.J. Mol. Cell. 2007; 27: 339-352Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The DEAD box helicases are grouped in helicase superfamily 2, whose members contain nine conserved motifs, including the Walker B motif DEAD from which they take their common name (Fig. 1). Escherichia coli contains five genes encoding for DEAD box proteins; csdA (formerly called deaD), dbpA, rhlB, rhlE, and srmB (4Iost I. Dreyfus M. Nucleic Acids Res. 2006; 34: 4189-4197Crossref PubMed Scopus (112) Google Scholar). The gene products have a common core of 350–400 amino acids, consisting of two domains that have the same fold found in the DNA-recombination protein RecA. Flanking this two-domain core are variable regions that are thought to be responsible for the differing properties and functions of the five helicases (4Iost I. Dreyfus M. Nucleic Acids Res. 2006; 34: 4189-4197Crossref PubMed Scopus (112) Google Scholar). DEAD box helicases can unwind short RNA duplexes that are no more than two helical turns in length (∼22 bp), and some helicases can displace avidly bound proteins from RNA (1Cordin O. Banroques J. Tanner N.K. Linder P. Gene (Amst.). 2006; 367: 17-37Crossref PubMed Scopus (702) Google Scholar, 5Jankowsky E. Fairman M.E. Curr. Opin. Struct. Biol. 2007; 17: 316-324Crossref PubMed Scopus (180) Google Scholar). A role of DEAD box RNA helicases as nonspecific RNA unfolding enzymes has been described (6Bhaskaran H. Russell R. Nature. 2007; 449: 1014-1018Crossref PubMed Scopus (83) Google Scholar). Yang and Jankowsky (7Yang Q. Jankowsky E. Nat. Struct. Mol. Biol. 2006; 13: 981-986Crossref PubMed Scopus (113) Google Scholar) have elegantly shown that DEAD box helicases use a single strand RNA region to facilitate loading onto duplex RNA where only a few base pairs are disrupted in an ATP-dependent manner, leading to destabilization of the remainder of the duplex and its spontaneous disassembly. Duplex unwinding by the RNA helicases differs from that of DNA or viral helicases, which in contrast act as highly processive translocases that may peel off many hundreds of complementary RNA and DNA strands in their paths (5Jankowsky E. Fairman M.E. Curr. Opin. Struct. Biol. 2007; 17: 316-324Crossref PubMed Scopus (180) Google Scholar). The crystal structure of the Drosophila DEAD box helicase Vasa in complex with a non-hydrolyzable ATP analogue and single-stranded RNA lends support to the proposed mechanism of duplex unwinding by the DEAD box family (8Sengoku T. Nureki O. Nakamura A. Kobayashi S. Yokoyama S. Cell. 2006; 125: 287-300Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). This complex structure shows that ATP binding promotes relative movement of the two RecA-like domains to induce a closed form of the helicase. The adoption of the closed conformation organizes the catalytic site and so enables hydrolysis of the terminal phosphate group of the ATP. The marked bending of the single-stranded RNA is suggestive of a mechanism that could physically disrupt a few RNA basepairs in a duplex (5Jankowsky E. Fairman M.E. Curr. Opin. Struct. Biol. 2007; 17: 316-324Crossref PubMed Scopus (180) Google Scholar, 8Sengoku T. Nureki O. Nakamura A. Kobayashi S. Yokoyama S. Cell. 2006; 125: 287-300Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). RNA helicases are often part of large macromolecular assemblies, and it is becoming increasingly apparent that their ATPase and/or RNA helicase activities can be modulated by the interactions that are formed within these complexes (3Bleichert F. Baserga S.J. Mol. Cell. 2007; 27: 339-352Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 9Alcazar-Roman A.R. Tran E.J. Guo S. Wente S.R. Nat. Cell Biol. 2006; 8: 711-716Crossref PubMed Scopus (215) Google Scholar, 10Carpousis A.J. Annu. Rev. Microbiol. 2007; 61: 71-87Crossref PubMed Scopus (357) Google Scholar, 11Rogers Jr., G.W. Richter N.J. Lima W.F. Merrick W.C. J. Biol. Chem. 2001; 276: 30914-30922Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 12Weirich C.S. Erzberger J.P. Flick J.S. Berger J.M. Thorner J. Weis K. Nat. Cell Biol. 2006; 8: 668-676Crossref PubMed Scopus (201) Google Scholar). One such example is the exon-junction complex, in which the ATPase activity of the DEAD box helicase component (eIF4AIII) is impeded by interaction with RNA and partner regulatory proteins (13Andersen C.B. Ballut L. Johansen J.S. Chamieh H. Nielsen K.H. Oliveira C.L. Pedersen J.S. Seraphin B. Le Hir H. Andersen G.R. Science. 2006; 313: 1968-1972Crossref PubMed Scopus (306) Google Scholar, 14Bono F. Ebert J. Lorentzen E. Conti E. Cell. 2006; 126: 713-725Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Turning to examples of bacterial helicase complexes, the E. coli DEAD box ATPase RhlB forms part of the multiprotein assembly known as the RNA degradosome. This large complex also comprises the endoribonuclease, RNase E, the exoribonuclease, polynucleotide phosphorylase, and the glycolytic enzyme, enolase. The co-localization of these enzymes in the degradosome allows cooperation of their activities (15Braun F. Hajnsdorf E. Regnier P. Mol. Microbiol. 1996; 19: 997-1005Crossref PubMed Scopus (43) Google Scholar, 16Cohen S.N. Cell. 1995; 80: 829-832Abstract Full Text PDF PubMed Scopus (105) Google Scholar). The presence of RhlB in the degradosome has been shown to facilitate RNA degradation by RNase E (17Khemici V. Poljak L. Toesca I. Carpousis A.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6913-6918Crossref PubMed Scopus (54) Google Scholar) and polynucleotide phosphorylase (18Coburn G.A. Miao X. Briant D.J. Mackie G.A. Genes Dev. 1999; 13: 2594-2603Crossref PubMed Scopus (150) Google Scholar, 19Khemici V. Toesca I. Poljak L. Vanzo N.F. Carpousis A.J. Mol. Microbiol. 2004; 54: 1422-1430Crossref PubMed Scopus (91) Google Scholar, 20Py B. Higgins C.F. Krisch H.M. Carpousis A.J. Nature. 1996; 381: 169-172Crossref PubMed Scopus (469) Google Scholar). This facilitation is presumably due to the removal of secondary structure from the substrate RNA (20Py B. Higgins C.F. Krisch H.M. Carpousis A.J. Nature. 1996; 381: 169-172Crossref PubMed Scopus (469) Google Scholar). RhlB interacts with the non-catalytic C-terminal “scaffold” domain of RNase E, which has also been shown to be required for polynucleotide phosphorylase and enolase binding (21Vanzo N.F. Li Y.S. Py B. Blum E. Higgins C.F. Raynal L.C. Krisch H.M. Carpousis A.J. Genes Dev. 1998; 12: 2770-2781Crossref PubMed Scopus (271) Google Scholar). The C-terminal domain is predicted to be predominantly unstructured, with some small regions of propensity for secondary structure being restricted to specific sites of recognition (22Callaghan A.J. Aurikko J.P. Ilag L.L. Gunter Grossmann J. Chandran V. Kuhnel K. Poljak L. Carpousis A.J. Robinson C.V. Symmons M.F. Luisi B.F. J. Mol. Biol. 2004; 340: 965-979Crossref PubMed Scopus (132) Google Scholar). The site of interaction on RNase E for RhlB has been identified to lie between residues 698 and 762 (23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). This engages the C-terminal RecA-like domain of the helicase (24Liou G.G. Chang H.Y. Lin C.S. Lin-Chao S. J. Biol. Chem. 2002; 277: 41157-41162Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and the binding surface has been proposed to encompass residues 368 to 397, which are not part of a conserved helicase signature motif (23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). From a homology model of RhlB based on the Drosophila Vasa structure this binding site is at a comparatively large distance of around 20 Å from the ATP binding pocket (Fig. 1). Consequently, the binding of RNase E must have an indirect effect on the ATPase activity of RhlB. Predictive methods have identified an exposed coil between residues 377 and 383 (Fig. 1) as a putative binding site (23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). This site is similar to the surface used in the eIF4AIII DEAD box helicase, with its partner proteins in the exon junction complex (13Andersen C.B. Ballut L. Johansen J.S. Chamieh H. Nielsen K.H. Oliveira C.L. Pedersen J.S. Seraphin B. Le Hir H. Andersen G.R. Science. 2006; 313: 1968-1972Crossref PubMed Scopus (306) Google Scholar, 14Bono F. Ebert J. Lorentzen E. Conti E. Cell. 2006; 126: 713-725Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). RhlB is the only E. coli DEAD box protein that requires a protein partner to stimulate its ATPase activity (10Carpousis A.J. Annu. Rev. Microbiol. 2007; 61: 71-87Crossref PubMed Scopus (357) Google Scholar, 25Bizebard T. Ferlenghi I. Iost I. Dreyfus M. Biochemistry. 2004; 43: 7857-7866Crossref PubMed Scopus (100) Google Scholar, 26Turner A.M. Love C.F. Alexander R.W. Jones P.G. J. Bacteriol. 2007; 189: 2769-2776Crossref PubMed Scopus (36) Google Scholar). The stimulating effect of RNase E on RhlB does not require the RNA-binding sites of the ribonuclease that flank the helicase interaction site (18Coburn G.A. Miao X. Briant D.J. Mackie G.A. Genes Dev. 1999; 13: 2594-2603Crossref PubMed Scopus (150) Google Scholar), suggesting that the boost of ATPase activity is not caused indirectly by increased recruitment of RNA. Indeed, we observe that the RNase E/RhlB interaction actually decreases the RNA affinity of the helicase. 4J. A. R. Worrall and B. F. Luisi, unpublished results.4J. A. R. Worrall and B. F. Luisi, unpublished results. Instead, the activation is likely to involve induced conformational changes in the RhlB, but the nature of these changes is not presently clear. Chandran et al. (23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar) have identified a distinctive sequence difference in one of the nine conserved motifs that may account for the activation of RhlB. The terminal amino acid of motif V is an Asp in all E. coli DEAD box proteins but is replaced in RhlB with a His (Fig. 1). Notably, residues in the predicted binding region for RNase E are co-conserved together with the motif V His residue, suggesting a link between RNase E binding on the one hand and the activation of ATPase activity on the other (23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). This link is explored as part of the present work. Our results confirm that there is communication between the ATPase catalytic site and the putative RNase E binding site; however, they also indicate that the activating effect of RNase E binding on ATPase activity cannot be ascribed to a few surface and active-site residues. Instead, the communication requires a constellation of amino acids that collectively affect the active site pocket. In the course of designing an accommodating cavity in the ATP binding pocket, we inadvertently discovered that substitution of residue Phe-10 near the adenine ring boosted ATPase activity tremendously. However, no activity could be detected in the presence of RNA, indicating that RNA binding has locked the enzyme into an inactive state. We discuss the implications for understanding how the enzyme might transduce energy from ATP hydrolysis into mechanical work. RhlB Expression and Site-directed Mutagenesis—Expression systems for three E. coli RNA helicases, RhlB, RhlE, and SrmB, were provided by A. J. Carpousis, CNRS Toulouse, France. The recombinant helicase genes are inserted within the multiple cloning site of a Novagen pET11a (Ampr) vector under the control of the T7 promoter. Mutations in each of the recombinant genes were created using a procedure based on the Stratagene QuikChange mutagenesis kit (27Braman J. Papworth C. Greener A. Methods Mol. Biol. 1996; 57: 31-44PubMed Google Scholar). The forward and reverse primers used to introduce the respective mutations in the RNA helicases are listed in Table 1. All clones were sequenced to corroborate that the intended mutations were successfully introduced. Site-directed variants of RhlB were also constructed in the di-cistronic pRneRhlB vector (see below) using the mutagenic primers reported in Table 1. Expression and purification was the same as outlined above for the wild-type complex.TABLE 1Mutagenic primer pairs used to generate the various mutations in the recombinant RNA helicases In lower case and underlined are the nucleotides that are changed from the original sequence to generate the respective helicase mutation.MutationMutagenic primer pairsRhlB F10A5′–CAGAACAGAAGgcTTCCGACTTCGCCC–3′5′–GGGCGAAGTCGGAAgcCTTCTGTTCTG–3′ F10M5′–CAGAACAGAAGaTgTCCGACTTCGCCC–3′5′–GGGCGAAGTCGGAcAtCTTCTGTTCTG–3′ H320D5′–GCCGCGCGTGGTTTGUgATATTCCGGCAGTGACG–3′5′–GCGTCACTGCCGGAATATcCAAACCACGCGCGG–3′ S381A5′–GGTCACTCAATTCCGGTAgcCAAATACAATCCGGACG–3′5′–CGTCCGGATTGTATTTGgcTACCGGAATTGAGTGACC–3′ Y383A5′–CCGGTAAGCAAAgcCAATCCGGACGCATTG–3′5′–CAATGCGTCCGGATTGgcTTTGCTTACCGG–3′RhlE D310H5′–CGCTGCGCGCGGCCTGcATATTGAAGAGCTGC–3′5′–GCAGCTCTTGAATATgCAGGCCGCGCGCAGCG–3′SrmB D313H5′–GCCGCGCGCGGTATCcACATTCCTGACGTCAG–3′5′–CTGACGTCAGGAATGTgGATACCGCGCGCGGC–3′ Open table in a new tab Recombinant Expression and Purification of RNA Helicases and Variants—Wild-type (WT) 5The abbreviations used are:WTwild typeADP-PNP5′-adenylyl-β,γ-imidodiphosphateITCisothermal titration calorimetryNi-NTAnickel-nitrilotriacetic acid. and site-directed variants of RhlB, RhlE, and SrmB were expressed in E. coli strain BL21(DE3) and isolated and purified as previously described (22Callaghan A.J. Aurikko J.P. Ilag L.L. Gunter Grossmann J. Chandran V. Kuhnel K. Poljak L. Carpousis A.J. Robinson C.V. Symmons M.F. Luisi B.F. J. Mol. Biol. 2004; 340: 965-979Crossref PubMed Scopus (132) Google Scholar, 23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). A final purification step was introduced to the published procedure involving S200 size exclusion chromatography. wild type 5′-adenylyl-β,γ-imidodiphosphate isothermal titration calorimetry nickel-nitrilotriacetic acid. Construction and Expression of the RNase E-(696–762)/RhlB Co-expression Vector—The Novagen pRSF-Duet-1 (Kanr) di-cistronic vector was used to construct a co-expression system for the complex formed between RhlB and residues 696–762 of RNase E. The vector consists of two multiple cloning sites each under the control of a T7 promoter. Primers for polymerase chain reaction (PCR) amplification were designed with restriction enzymes sites added for facile cloning of the products into the multiple cloning sites of the pRSF vector. The rhlB gene was amplified form the pET11a vector and ligated in the upstream NdeI and downstream XhoI sites in the second pRSF multiple cloning site. DNA encoding for amino acid residues 696–762 of RNase E was amplified from the full-length RNase E in a pET15b vector. The product was cloned in the upstream BamHI and downstream SalI sites of the first pRSF multiple cloning site creating a His6 tag at the N terminus. Several clones were evaluated by restriction digests and sequencing in both multiple cloning sites to corroborate the correct insertion of the amplified genes into the vector. The final vector was named pRneRhlB. A C-terminal truncation of RhlB was constructed in the pRneRhlB vector. The codon (CTC) for amino acid 398 (Leu) in RhlB was replaced by a stop codon (TAA) using the QuikChange strategy. The final clone was designated pRneRhlBΔ1–397. E. coli BL21(DE3) cells were transformed with pRneRhlB or pRneRhlBΔ1–397 and single transformants were transferred to 50 ml of 2× YT medium containing 50 μg liter-1 kan and incubated overnight at 37 °C. Two-liter Erlenmeyer flasks containing 500 ml of 2× YT medium and 50 μg liter-1 kan were inoculated with 5 ml of overnight culture and incubated at 37 °C until an A600 of 0.5–0.6 was reached. At this point expression of the recombinant genes was induced with 1 mm isopropyl β-d-thiogalactopyranoside. After 3 h the cells were harvested and resuspended in lysis buffer (50 mm Tris/HCl, pH 8.0, 200 mm NaCl, 100 mm KCl, 5 mm MgCl2, 5 mm imidazole, and an EDTA-free protease inhibitor mixture tablet) and passed three times through an Emulsiflex-05 cell disruptor (Avestin). The soluble fraction was collected by centrifugation (35,000 × g, 4 °C) and loaded to a Ni-NTA Hitrap column (GE Healthcare). Extensive washing with lysis buffer was followed by a gradient elution with lysis buffer supplemented with 500 mm imidazole (buffer B). Two peaks eluted at 20 and 40% buffer B and fractions were analyzed by SDS-PAGE electrophoresis. The first peak contained RhlB and the second peak was enriched with RhlB and the His6-tagged RNase E-(696–762). Fractions from the second peak were pooled and dialyzed against 50 mm Tris/HCl, pH 8.0, and 50 mm NaCl (buffer C) and loaded to an SP column (GE Healthcare) equilibrated in buffer C. A linear gradient (0–100% buffer C containing 1 m NaCl) was applied and a major peak eluted at ∼350 mm NaCl. Fractions were pooled, concentrated in 30-kDa cut-off Centricon units (Vivascience), and loaded to an S200 size exclusion column (GE Healthcare). A major peak eluting at a column volume corresponding to a species with a molecular mass of ∼50 kDa was obtained. Analysis of this peak by SDS-PAGE electrophoresis revealed it to contain the complex between RhlB and RNase E-(696–762). Construction and Purification of RNase E-(696–762) Peptide—A plasmid with the DNA sequence for overexpression of RNase E residues 696–762 was created. The PCR-amplified DNA fragment of RNase E-(696–762) was ligated into the upstream BamHI and downstream SalI sites in the first multiple cloning site of the pRSF-Duet-1 vector. Overexpression in 2× YT medium supplemented with 50 μg liter-1 yielded a protein product with an N-terminal His6 tag that eluted from a Ni-NTA column as a broad peak. Fractions analyzed by SDS-PAGE electrophoresis showed a major band running at ∼18 kDa. These fractions were pooled, concentrated, and applied to an S200 size exclusion column. Isothermal Titration Calorimetry—Protein samples for isothermal titration calorimetry (ITC) analysis were dialyzed extensively against 50 mm potassium phosphate, pH 7.4, 100 mm NaCl, and 1 mm dithiothreitol for 24 h at 4 °C. After dialysis samples were concentrated using either 30- or 5-kDa cut-off Centricon units (Vivascience) and concentrations were determined from UV spectroscopy using extinction coefficients (ϵ) at 280 nm of 36,120 m-1 cm-1 for RhlB and variants, and 1,490 m-1 cm-1 for RNase E-(696–762). RNase E-(696–762) solutions of 300 μm were placed into the syringe and titrated into the sample cell containing 20 μm WT RhlB or a site-directed variant with stirring at 310 rpm during the experiment. All titration experiments were performed at 25 ± 0.1 °C on a Microcal VP-ITC calorimeter with an injection volume of 2 μl for the first and 5 μl for all subsequent titration points, 60-s initial equilibrium delay and 270-s pause between injections. Binding isotherms were analyzed with Microcal Origin 7.0. In each case the first data point was discarded and the baseline adjusted manually. The integrated data were corrected for the heat of dilution of RNase E-(696–762) into the buffer and binding isotherms were analyzed with 1:1 and 2:1 binding models using the software package of the manufacturer (28Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2386) Google Scholar). Nanospray Mass Spectrometry—The “apo”-RhlB (in the absence of the RNase E peptide) and the co-expressed complex, after size exclusion chromatography, were prepared to concentrations between 5 and 20 μm and buffer exchanged into 250 mm ammonium acetate (Micro Bio-Spin chromatography columns, Bio-Rad). All spectra were acquired on a Q-ToF 2 modified for high mass operation and equipped with a Z-spray nanoflow source (Waters, Manchester, UK) (29Sobott F. Hernandez H. McCammon M.G. Tito M.A. Robinson C.V. Anal. Chem. 2002; 74: 1402-1407Crossref PubMed Scopus (422) Google Scholar). The following experimental parameters were used: capillary voltage 1.7 kV, cone voltage 80–120 V, cone gas 100 liter h-1, collision cell voltage up to 200 V, ion transfer stage pressure 4.0 × 10-3 to 2.0 × 10-2 mbar, argon collision gas at a collision cell pressure of 2–7 μbar. External calibration was achieved by using a 33 mg ml-1 aqueous solution of cesium iodide (Sigma). Calibration, acquisition, and processing were carried out using MassLynx software (Waters, Manchester, UK). ATPase Assays—ATPase activity of RNA helicases was monitored spectrophotometrically on a Shimadzu BioSpec-1601 UV-visible spectrophotometer thermostatted at 25 ± 0.1 °C using the Molecular Probes EnzCheck Phosphate Assay kit (Invitrogen). The assay is based on a method originally described by Webb (30Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Crossref PubMed Scopus (468) Google Scholar). In the presence of inorganic phosphate (Pi) released by hydrolysis of ATP, the substrate 2-amino-6-mercapto-7-methylpurine riboside is converted by purine nucleoside phosphorylase (PNP) to ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine. This enzymatic conversion is accompanied by a shift in the maximum absorbance from 330 nm for the substrate to 360 nm for the product. Assays were performed in 1-ml volumes in 50 mm Tris/HCl, pH 7.5, and supplemented with 200 μm ATP and 400 μm MgCl2. Bakers' yeast RNA (Sigma), when included, was at a final concentration of 40 μg/ml. Reaction components were preincubated for 10 min at room temperature before the reaction was started by the addition of the desired helicase or helicase-RNase E complex to a final concentration of 2.4 μm. Reactions were monitored for 300 s with initial rates calculated from the rate curve over the first 30 s converted into the amount of phosphate released/min/mol of protein from a Pi standard curve. Reported rates are an average of two or three independent experiments. Site-directed Mutagenesis of Three E. coli DEAD Box Proteins and the Co-expression of an RNase E·RhlB Complex—Site-directed variants of three E. coli RNA DEAD Box helicases, RhlB, RhlE, and SrmB were constructed (Table 1). The final expression constructs produced proteins that were highly overexpressed in E. coli, were entirely soluble, and could be purified to >95% as judged from SDS-PAGE. From previous studies it was found that RhlB forms a complex with RNase E (19Khemici V. Toesca I. Poljak L. Vanzo N.F. Carpousis A.J. Mol. Microbiol. 2004; 54: 1422-1430Crossref PubMed Scopus (91) Google Scholar, 21Vanzo N.F. Li Y.S. Py B. Blum E. Higgins C.F. Raynal L.C. Krisch H.M. Carpousis A.J. Genes Dev. 1998; 12: 2770-2781Crossref PubMed Scopus (271) Google Scholar, 22Callaghan A.J. Aurikko J.P. Ilag L.L. Gunter Grossmann J. Chandran V. Kuhnel K. Poljak L. Carpousis A.J. Robinson C.V. Symmons M.F. Luisi B.F. J. Mol. Biol. 2004; 340: 965-979Crossref PubMed Scopus (132) Google Scholar, 23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). To aid in investigating the properties of this complex, a di-cistronic expression vector was constructed encoding WT RhlB or its site-directed variants and an N-terminal His6-tagged 67-amino acid peptide of RNase E corresponding to residues 696–762. Elution from a Ni-NTA column followed by S200 size exclusion chromatography revealed the RNase E peptide to remain bound to RhlB, indicating that the interaction is avid (Fig. 2). The normalized intensities of the bands on a denaturing gel suggest a 1:1 stoichiometry, corrected for dye uptake in proportion to the molecular mass. It is noted that the Coomassie-stained band for the RNase E peptide runs at twice the apparent molecular mass (∼18 kDa) than the predicted 9163 Da, which includes the 67 amino acids corresponding to RNase E residues 696–762 and 13 additional amino acids coming from the plasmid of which six are the N-terminal His tag. Non-dissociative mass spectrometry analysis provides a stoichiometry of 1:1 and a molecular mass of the RNase E peptide of 9189 Da (Fig. 2). The binding of one RNase E to one RhlB is in accord with earlier results using the R-domain construct of RNase E (residues 628–843), which includes the two putative RNA binding domains flanking the RhlB binding site (Fig. 2) (22Callaghan A.J. Aurikko J.P. Ilag L.L. Gunter Grossmann J. Chandran V. Kuhnel K. Poljak L. Carpousis A.J. Robinson C.V. Symmons M.F. Luisi B.F. J. Mol. Biol. 2004; 340: 965-979Crossref PubMed Scopus (132) Google Scholar, 23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). Analysis of Interactions of RhlB Surface Mutants with RNase E—Residues Ser-381 and Tyr-383 of RhlB were predicted previously to be important for binding/recognition of RNase E (23Chandran V. Poljak L. Vanzo N.F. Leroy A. Miguel R.N. Fernandez-Recio J. Parkinson J. Burns C. Carpousis A.J. Luisi B.F. J. Mol. Biol. 2007; 367: 113-132Crossref PubMed Scopus (58) Google Scholar). To test this, the S381A and Y383A mutants of RhlB were overexpressed and purified. An expression plasmid to overexpress N-terminal His6-tagged RNase E, residues 696–762, was also constructed. The peptide was analyzed by SDS-PAGE and it migrates with the same apparent size as seen
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