Biochemical and Biophysical Research Communications 408 (2011) 459–464

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Biochemical and Biophysical Research Communications

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Deciphering the catalytic machinery in a universally conserved ribosomebinding ATPase YchF

Sushil Kumar Tomar, Prashant Kumar, Balaji Prakash ⇑Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208 016, India

a r t i c l e i n f o

Article history:Received 1 April 2011Available online 19 April 2011

Keywords:YchF ATPasePotassium dependent stimulationRibosome binding ATPase70S/50S bindingConserved bacterial GTPase

0006-291X/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.bbrc.2011.04.052

⇑ Corresponding author. Fax: +91 512 2594010.E-mail address: bprakash@iitk.ac.in (B. Prakash).

a b s t r a c t

YchF, a universally conserved protein, hitherto thought to be a GTPase, was shown to be an ATPase basedon structural and biochemical studies on hOLA1, a human ortholog of YchF. However, the cellular role ofYchF is unclear. Based on the presence of a RNA binding domain in this protein and significant homologyto ribosome binding Obg family GTPases, we examined its ability to associate with the ribosome. Here,we show that Escherichia coli YchF binds the 50S and 70S ribosomal particles in a nucleotide independentmanner and it hydrolyzes ATP utilizing a potassium dependent mechanism. A potassium mediated accel-eration of hydrolysis activity was thus far known for a few GTPases. Like these, YchF too conserves thestructural features required for K+ coordination, making it a unique ribosome binding ATPase utilizinga similar mechanism. Furthermore, we show that Lys78 is an important determinant of the potassiumdependent ATPase activity.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

GTPases are universally conserved regulatory proteins that reg-ulate several important biological processes ranging from signaltransduction, intercellular transport, protein synthesis, ribosomebiogenesis and so on. Using a set of conserved motifs G1–G4, theybind guanine nucleotides and cycle between an active GTP bound‘‘ON’’ state to activate a downstream molecule in a signalingcascade and upon hydrolysis of GTP, achieve an inactive GDPbound ‘‘OFF’’ state [1]. Interestingly, bacteria require a minimumset of 11 GTPases, also known as universally conserved GTPases,essential for their survival [2]. Among these Ffh and FtsY (alsoknown as secretion factors) are required for co-translational tar-geting of nascent polypeptide chains to the plasma membrane[3]. EF-G, EF-Tu and IF-2 are involved in protein synthesis [4].Era binds to 30S ribosomal subunit and is essential for processing16S rRNA [5,6]. YihA binds to 50S ribosomal subunit and has beenshown to be important for its assembly [7]. EngA and Obg bind toboth 50S and 30S ribosomal subunits and like YihA, have beenshown to participate in the assembly of 50S [8,9]. LepA helps inback translocation of the ribosome and participate in proofreadingfunction during protein synthesis [10]. MnmE is required for tRNAmodification [11].

YchF belongs to the Obg family of GTPases. It is not only con-served in prokaryotes but also found in other kingdoms of life.Studies on the human ortholog of YchF, known as hOLA1 (human

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Obg like ATPase), surprisingly showed that it preferentially hydro-lyzes ATP over GTP [12]. Crystal structures of YchF from H. influ-enza and hOLA1 show that the polypeptide chain is folded intothree discontinuous domains – a typical NTPase domain at the Nterminus, followed by a a-helical coiled coil domain and a TGS likeRNA binding domain at the C-terminus [12,13]. In Escherichia coli,ychF is co-transcribed with another gene named pth [14]. Interest-ingly, similar to YchF, pth is also highly conserved and found in allorganisms; it codes for peptidyl-tRNA hydrolase (Pth), which is re-quired in ribosome recycling [15]. Despite the available studies, theprecise biological role of YchF is still not clear. It was hithertothought to belong to a subset of GTPases, known as HAS-GTPases(Hydrophobic Amino acid Substituted for catalytic Glutamine –GTPases), based on conserved sequence motifs. These GTPases,despite possessing a hydrophobic residue in place of a catalyticglutamine (Gln 61 in Ras) conserved in most GTPases, are capableof hydrolyzing GTP [16]; whereas a similar substitution in Rasshows complete loss of hydrolysis activity and the mutation isknown to be oncogenic [17]. Earlier, we proposed that, unlike inclassical GTPases, a catalytic residue in HAS-GTPases could be sup-plied from regions other than the G3 motif (Switch II) [16]. InMnmE, a catalytic residue is provide by an a-helix, adjacent tothe G3 (DxxG) motif and GTP hydrolysis is stimulated by a potas-sium ion. The K+ ion is stabilized by a region named the K-loop (apart of G2 region) and a conserved Asn from the G1 motif (or the P-loop) [11]. A similar K+ dependent hydrolysis mechanism is alsoemployed by GTPases, YqeH and FeoB [18,19]. More commonly,GTP hydrolysis is accelerated due to an arginine residue (argininefinger) supplied by a regulatory protein – GTPase Activating

460 S.K. Tomar et al. / Biochemical and Biophysical Research Communications 408 (2011) 459–464

Protein (GAP), into the active site of the GTPase. This arginine neu-tralizes the developing negative charge on the nucleotide duringhydrolysis [20]. In MnmE, YqeH and FeoB, K+ plays a similar role[11,18,19]. Based on the fact that YchF possesses a K-loop and aconserved Asn in the P-loop (similarly as MnmE, YqeH and FeoB),we investigated K+ mediated ATP hydrolysis by YchF. Indeed, YchFutilizes K+ for ATP hydrolysis and a residue, Lys78, plays a key rolein this activity. Further, since YchF conserves features common tothe ribosome binding Obg family GTPases [13], we inquired if itbinds ribosome. We find that YchF binds 50S and 70S ribosomesin a nucleotide independent manner.

2. Materials and methods

2.1. Plasmids and proteins

YchF full length gene was amplified from E. coli K12 genomicDNA using forward (50-GCCATATGATGGGATTCAAATGCG-30) andreverse (50-CGAAGCTTTTAATGGTGATGGTGATGGTGATGGACGTTG-AAAAGG-30) primers, respectively. This was cloned into a modifiedpQE2 vector between NdeI and HindIII restriction sites to generate aplasmid expressing a C-terminal His-tagged protein. All themutants N13A, K78A and K78R were generated by overlap PCRtechnique, using full length YchF as template with 50-ATCGTCGGTTTGCCCGCCGTCGGGAAATCTACC-30, 50-GATATCGCCG-GTCTGGTAGCAGGCGCATCGAAAGGCG-30 and50-GATATCGCCGGTC-TGGTAAGAGGCGCATCGAAAGGCG-30primers,respectively.Proteinswere over-expressed in E. coli DH5a cells using 0.1 mM isopropyl-b-D-1-thiogalacto-pyranoside (IPTG). Cells were harvested and lysed inphosphatebuffer(50 mM)pH7.4,150 mMNaClandHis-cocktailpro-tease inhibitor (Sigma–Aldrich). Lysates were treated with mild con-centrations of DNase and RNase and supernatants were obtained bycentrifuging the lysates at 30,000g for 1 h. These were loaded onto aNi–NTA column (Histrap-Amersham) pre-equilibrated with phos-phate buffer. Non-specific proteins bound to the column werewashed and eluted in same buffer using 0–500 mM gradient of imid-azole. Fractions containing the protein were further subjected to sizeexclusionchromatographyonasuperdex200column(Amersham)in50 mM Tris–HCl buffer (pH 8.0).

2.2. Nucleotide hydrolysis assays

Nucleotide hydrolysis assays were carried out at 37 �C for45 min in 10 ll reaction volumes. These contains 5 lM enzymein hydrolysis reaction buffer (50 mM Tris–HCl, pH 8.0, 1 mM DTT,5 mM MgCl2), 100 lM of cold nucleotide (GTP or ATP), anda[32P]GTP or a[32P]ATP in presence of 200 mM of the salt indicatedin the figures. The reaction was stopped by adding 2 ll of 6 M for-mic acid and denatured protein was precipitated by centrifugationat 13,000 rpm for 10 min. About 4 ll of the supernatant were spot-ted on PEI-coated TLC (Merck) plate to resolve a[32P] labeleddiphosphate and unutilized triphosphate species in 0.75 M KH2PO4

buffer (pH 3.4). TLC Plates were then subjected for autoradiogra-phy and spots corresponding to the nucleotides were cored out.These were then subjected to quantification using a scintillationcounter (Perkin-Elmer Life Sciences). Values of the intrinsic hydro-lysis of GTP/ATP, were negated from the aforesaid measurementsand specific activity (amount of Pi released for a given concentra-tion of enzyme for a certain time (min)) of the enzyme wascalculated. All the reactions were setup in duplicates.

2.3. Ribosome binding studies

Crude ribosomes from E. coli DH5a cells were purified and storedat �80 �C, as described before [9]. Ribosome co-sedimentation

experiment was performed using purified components i.e.250 pmol of protein and 5 A254 units of the crude ribosome. Thesewere incubated in ribosome binding buffer (50 mM Tris–HCl, pH7.5, 10 mM MgCl2 and 50 mM NH4Cl) in presence of appropriatenucleotides [ADP or AMPPNP (Sigma)] at 2 mM concentrations.Sample mixtures were incubated for 30 min at 37 �C and were thenloaded on a 17 ml continuous sucrose gradient (15–43%) preparedin 20 mM Tris–HCl, pH 7.5, 6 mM MgCl2 and 50 mM NH4Cl. Thesewere subjected to ultra centrifugation in Sorvall Sure-Spin 630 ro-tor (28,000 rpm) for 10 h. Using upward displacement method, 30equal fractions were collected while monitoring absorbance at254 nm. These fractions were analyzed for the presence of 16Sand 23S rRNA on a formaldehyde agarose gel. Only the peak frac-tions containing 30S, 50S and 70S were subjected to SDS–PAGEanalysis followed by immuno-blotting using anti-His antibodiesto detect His-YchF protein.

2.4. Fluorescent nucleotide binding assays

Fluorescent nucleotide binding studies were carried out as de-scribed before [9]. Mant (N-methyl-30-O-anthranoyl) nucleotides(0.5 lM) and proteins (5.0 lM) were incubated at room tempera-ture in 50 mM Tris–HCl buffer (pH 8.0) containing 5 mM MgCl2.Fluorescence was monitored with an excitation and emissionwavelength of 355 and 400–600 nm, respectively. For transitionstate analysis (Fig. 3) mixture(s) were made in similar conditionswith addition of 200 mM salt (NaCl/KCl/NH4Cl/RbCl/CsCl), 0.5 lMAlCl3 and 5 lM NaF.

2.5. Analytical gel filtration

Analytical gel filtration experiments were performed in equili-bration buffer containing 50 mM Tris, pH 8.0, 5 mM MgCl2 and200 mM KCl with 150 lM YchF protein for the nucleotide-freestate. For the nucleotide bound states, reaction mixtures were pre-pared in similar buffer conditions with the addition of 0.5 mM ADPor 0.5 mM ADP and 0.5 mM AlCl3, 5 mM NaF (for the transitionstate analysis). These were incubated at room temperature for15 min and then 100 ll of reaction mixture was loaded onto aSuperdex 200 5/150 GL column (Amersham Biosciences). Chro-matograms were recorded at 280 nm.

3. Results

3.1. E. coli YchF is an ATPase that binds to 50S and 70S in a nucleotideindependent manner

YchF shares significant sequence similarity with the Obg familyof GTPases that hydrolyze GTP. Intriguingly, the human ortholog ofYchF, hOLA1, was shown to hydrolyze ATP more efficiently thanGTP [12]. Hence, we inquired whether E. coli YchF too, would pref-erentially hydrolyze ATP. For this and all the studies carried outhere, C-terminal His-tagged YchF (termed YchF throughout) wasprepared. Radioactively labeled nucleotides were employed inATP and GTP hydrolysis assays in presence of 200 mM NaCl, asdescribed in ‘‘Section 2’’. Fig. 1A shows that E. coli YchF hydrolyzesATP (specific activity 0.20214 ± 0.01118 min�1) but not GTP (spe-cific activity 0.02288 ± 0.02327 min�1). This confirms that YchF isalso an ATPase, in accordance with the previous study on hOLA1[12].

Obg family of GTPases have a conserved RNA binding domainand are known to bind ribosomes. Therefore, we anticipated thatYchF too may bind the ribosome. To examine this, ribosomeco-purification experiments were performed. In these experi-ments, 250 pmol of YchF was incubated with 5 A254 units of crude

Fig. 1. E. coli YchF is an ATPase and binds 50S and 70S. GTP and ATP hydrolysis activities of YchF in presence of 200 mM NaCl were estimated from radioactively labelednucleotide hydrolysis assays (see ‘‘Section 2’’). Specific activities for GTP, ATP hydrolysis by YchF are represented as bar plots. Error bars represent standard deviations fromthe average (A). YchF-ribosome co-fractionation experiments were performed as described in ‘‘Section 2’’. Purified components, YchF and crude ribosomes from E. coli, wereincubated in presence of indicated nucleotides. Peak fraction corresponding to 30S, 50S and 70S were analyzed for the presence of the protein. His-YchF was detected byWestern blotting using an anti-His antibody. The ribosome profile indicating the various ribosomal subunits is shown. YchF co-fractionates with 50S and 70S (but not with30S) independent of the nucleotide bound state (B).

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ribosomes in presence or absence of nucleotides. These reactionmixtures were subjected to co-sedimentation experiments andribosome profiles were recorded at 254 nm. Presence of proteinin the peak fractions obtained following ultra centrifugation wasdetected by Western blotting using anti-His antibodies. A repre-sentative ribosome profile indicating various ribosomal subunitsis shown in Fig. 1B. Immuno-blots shown in Fig. 1B reveal thatYchF binds 50S and 70S (but not 30S), irrespective of the presenceor absence of nucleotides, ADP or AMPPNP (a non hydrolysableanalog of ATP).

Fig. 2. Monovalent cations accelerate the ATPase activity of YchF. Protein sequencescorresponding to G1 and G2 motifs were included in the alignment and shown here. ConSwitch-I are shown (A). Specific ATPase activity of wt YchF was calculated in presenceCl > NH4Cl > RbCl > NaCl > CsCl. The activity, in presence of NaCl and KCl, for N13A mutanexperiments indicate that the mutation N13A does not compromise nucleotide binding

3.2. ATP hydrolysis by YchF is accelerated by potassium ions

YchF displays slow intrinsic ATP hydrolysis (Fig. 1A). For severalGTPases, GAPs, by means of an ‘Arg finger’ are known to accelerateGTP hydrolysis rates [20]. However, an emerging theme is that therole of Arg finger is also accomplished by a potassium ion, as inMnmE, YqeH and FeoB. In these, a conserved Asn in the G1 motifand an insertion in switch-I, termed the K-loop, are important toposition a K+ ion that stabilizes the transition state structure, simi-larly as the Arg finger does. YchF reveals a high sequence similarity

of YchF, FeoB, MnmE and YqeH were aligned using ClustalX. Only the regionsserved and similar residues are marked by stars and dots, respectively. K-loop and

of indicated salts. A stimulation of this activity by these ions follows the order – Kt of YchF does not indicate a similar stimulation (B). Fluorescent mant-ADP binding

. Emission spectra for both wt and N13A, along with the controls are shown (C).

Fig. 3. YchF is a monomer irrespective of the nucleotide bound state. The influence of various salts on nucleotide binding was examined as in Fig. 2. Intrinsic fluorescence bymGDP (curve 2) is enhanced upon addition of YchF (curve 3). However, no further change in the fluorescence intensity is seen upon addition of AlFx and the indicated salts(curves 4–8) (A). Analytical gel filtration experiments for YchF were conducted in apo, in presence of ADP and ADP�AlFx. Absorbance at 280 nm is shown in milli-absorbanceunits (mAU). The elution profiles indicate that it remains a monomer irrespective of the bound nucleotide (B).

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with these proteins, particularly in the G1 and G2 motifs and con-serves the Asn (Asn13) in the G1 or P-loop region (Fig. 2A). Thisled us to investigate whether YchF too, shows an accelerated ATP-ase activity in the presence of potassium and other monovalent cat-ions. To verify this, ATP hydrolysis assays were performed inpresence of various salts. Fig. 2B shows that stimulation of ATPhydrolysis is maximal in presence of K+ (specific activity0.7979 ± 0.06888 min�1), followed by NHþ4 (specific activity0.78846 ± 0.08617 min�1), Rb+ (specific activity 0.63205 ± 0.05849min�1), Na+ (specific activity 0.20214 ± 0.01118 min�1) and Cs+

(specific activity 0.19872 ± 0.03678 min�1). This behavior is similarto the acceleration of hydrolysis rates noted for MnmE, YqeH andFeoB [11,18,19].

The Asn of the G1 motif is important to coordinate the cation, inthese enzymes. To examine whether Asn13 plays a similar role inYchF as well, it was mutated to Ala and ATP hydrolysis was mea-sured in presence of NaCl and KCl. Fig. 2B shows that N13A mutant,unlike the wild type, does not display differential stimulation ofATP hydrolysis, in presence of potassium (specific activity0.38254 ± 0.05258 min�1) or sodium ions (specific activity0.27355 ± 0.0627 min�1). However, the mutant N13A continuesto bind nucleotides similarly as the wild type protein, as verifiedfrom fluorescent nucleotide (mant-ADP) binding experiments(Fig. 2C).

3.3. YchF is monomeric irrespective of the nucleotide bound state

In MnmE, cation dependent stimulation of GTP hydrolysis leadsto dimerization of the protein, specifically in the transition state. Inthis, the catalytic pockets of the two monomers of MnmE face eachother and residues from switch I (G2 motif) make important inter-actions to stabilize the dimer interface [11]. Based on a significantsimilarity in the switch II regions of YchF and MnmE (Fig. 2A), wefurther asked whether a similar phenomenon, i.e. oligomerizationin presence of K+ ions, occurs in YchF too. This was investigatedby the use of fluorescent mant-nucleotide binding assays, wherethe transition state of YchF was achieved by incubating the proteinwith mant-ADP and AlFx together with various monovalentcations. In these, a significant change in hydrophobicity of thenucleotide binding pocket, for e.g. due to dimerization in case ofMnmE, would be reflected by an increased fluorescence emission.In case of MnmE, where dimerization occurs in presence of K+

but not Na+ ions, an increase in fluorescence intensity was reported

for KCl but not NaCl [11]. However, for YchF, a similar change influorescence intensity could not be observed in presence of K+ orany other cations (Fig. 3A), suggesting that no appreciabledifference in hydrophobicity of the nucleotide binding pocket oc-curs in the transition state.

Furthermore, YchF, in the absence of nucleotides or in presenceof ADP or in the transition state, was tested for variation inoligomeric state, if any, using analytical gel filtration experiments.Elution profiles suggest that, unlike as reported for MnmE [11],YchF remains a monomer in solution, irrespective of the nucleotidebound state (Fig. 3B).

3.4. K78 plays an important role in determining the potassiumdependent ATPase activity

YchF, albeit an ATPase exhibits features of HAS-GTPases, simi-larly as MnmE, YqeH and FeoB, by carrying a hydrophobic substi-tution in place of the conserved catalytic Glutamine of classicalGTPases [16]. The catalytic Gln in the classical GTPases is knownto abstract a proton from a water molecule, which then acts as anucleophile to trigger GTP hydrolysis. In MnmE and YqeH, it hasbeen shown that a catalytic residue that similarly activates a watermolecule, is provided from an a-helix, next to the G3 (DxxG) motif[11,18]. We therefore attempted to identify a potential catalyticresidue present in the analogous helical region of YchF. Sequenceanalysis reveals a conserved Lys/Arg (K78 in E. coli YchF) in mostYchF homologs (Supplementary Fig. S1). This residue is in thevicinity of the catalytic pocket and hence may serve a similar rolein activating a water molecule. To probe this, we generated alanineand arginine mutants of lysine78 in YchF and examined their abil-ity to hydrolyze ATP. Nucleotide binding by these mutants wasverified as before, using mant-ADP binding experiments (Fig. 4B).ATP hydrolysis activities of K78A and K78R are shown in Fig. 4A.Surprisingly, the mutant K78A, unlike the wild type, does not dif-ferentiate between Na+ (specific activity 0.73503 ± 0.02429 min�1)and K+ (specific activity 0.90937 ± 0.00612 min�1). On the otherhand, its activity in presence of Na+ is higher (specific activity0.73503 ± 0.02429 min�1) than the wt protein (specific activity0.20214 ± 0.01118 min�1). In contrast, the K78R mutant behaveslike the wt protein; it retains K+ dependent stimulation of ATPaseactivity and differentiates between K+ and Na+ (specific activityin NaCl 0.07522 ± 0.01976 min�1, specific activity in KCl0.37825 ± 0.07792 min�1). Therefore, contrary to its expected role

Fig. 4. K78 plays an important role in determining potassium dependent ATPase activity of YchF. Specific activities of K78A and K78R were estimated as in Fig. 1A. K78A doesnot show significant difference in activity when compared for NaCl and KCl. However, its activity is higher than that of the wt protein in presence of NaCl. Interestingly, K78Rbehaves like wt and retains K+ dependent stimulation of ATPase activity (A). Control mant-ADP binding experiments, were carried out, as in Fig. 2, to ensure that both mutantsbind the nucleotide (B).

S.K. Tomar et al. / Biochemical and Biophysical Research Communications 408 (2011) 459–464 463

as a catalytic residue, K78 seems to play a role in determining K+

dependent activation of ATP hydrolysis, in YchF.

4. Discussion

YchF is a highly conserved protein in all three kingdoms of life.However, its biological role has not been investigated. In this work,we attempt to characterize E. coli YchF and show that similar toother members of the Obg family, it also binds the ribosome (50Sand 70S) (Fig. 1A). However, this binding is independent of thebound nucleotide. Furthermore, binding to 70S hints at a possiblerole in translation. This possibility gains prominence, as the genepth that codes for peptidyl-tRNA hydrolase (Pth) is co-expressedas a bicistronic mRNA with YchF. Pth functions in tRNA recyclingfrom stalled ribosomes [14,15]. However, to examine this possibil-ity for YchF in translation warrants a detailed study.

Interestingly, in line with the observations for hOLA1, YchF, hit-erhto thought to be a GTPase, hydrolyzes ATP (Fig. 1A). Interest-ingly, the mechanism of potassium dependent stimulation ofhydrolysis activity in YchF appears to be a feature similar toGTPases MnmE, YqeH and FeoB [11,18,19]. ATP and GTP differ inthe base region of the nucleotide, but are similar at the site ofhydrolysis, i.e. the phosphates. Hence, it appears that YchF andthese GTPases, evolved distinct specificities for nucleotides whileretaining K+ dependent hydrolysis (Fig. 2B). These enzymes showmaximum acceleration in presence of K+, but other cations suchas NHþ4 and Rb+ are also capable of accelerating nucleotide hydro-lysis. This is because of the similar ionic radii of these ions (K+

138 pm, NHþ4 144 pm, Rb+ 152 pm), which enables them to substi-tute for the role of K+ in the catalytic pocket. On the contrary, Na+

or Cs+ have smaller or larger radii (Na+ 102 pm, Cs+ 167 pm), andcannot replace K+ to accelerate nucleotide hydrolysis. Comparedto concentrations of NHþ4 and Rb+, concentration of K+ is muchhigher in the cell and therefore it seems appropriate that K+

accelerates hydrolysis in these enzymes.Nucleotide binding and analytical gel filtration experiments

suggest that unlike MnmE, YchF remains monomeric irrespectiveof the bound nucleotide (Fig. 3B). In MnmE, it was reasoned thatR and D of the TTRD motif in the G2 motif (Switch-I) is responsiblefor dimerization. Perhaps the absence of these residues in YchF,where this motif is replaced by CTIE (indicated in Fig. 2A) explainsthe monomeric behavior. YchF may be categorized as a member of

HAS-GTPases that lack a catalytic Gln. We had earlier hypothesizedfor HAS-GTPases, that a catalytic residue could be provided from aregion other than the canonical Switch II (G3) region [16]. Indeed,this was verified by studies on MnmE and YqeH, where a catalyticresidue, E282 and D57, respectively, is provided by a a-helical re-gion adjacent to Switch II [11,18]. We reasoned that K78 in YchF,present in the analogous helical region, may similarly play a cata-lytic role; this residue corresponds to a Lys or an Arg in YchF homo-logs. Arg and Lys differ only in the length of the side chain andcould activate a water molecule to act as catalytic residues. Toour surprise, YchF-K78A mutant shows similar hydrolysis activitiesin presence of Na+ or K+ and unlike the wt protein, does not distin-guish between the two. However, the activity is higher than that ofthe wt protein in presence of Na+ (Fig. 4A). On the contrary, K78Rretained potassium specific stimulation of ATPase activity like thewt. This explains the presence of an Arg or a Lys at this positionamong YchF homologs. Overall, it appears that K78 does not playa catalytic role as anticipated, but is essential in determining K+

dependent hydrolysis in YchF. Further, K+ dependent stimulationmight be an additional regulation for YchF to satisfy its cellularfunction. Structural studies in the transition state would revealmechanistic details of this regulation. In most of the ribosomebinding GTPases, ribosome binding is achieved in GTP bound stateand upon hydrolysis to GDP, they dissociate from the ribosome.How is the association and dissociation of YchF-ribosome complexregulated, since its binding to 70S is independent of its nucleotidebound state? Answering this question and to further understandthe role of this conserved ribosome binding ATPase in the cell,warrants rigorous experimentation.

Acknowledgments

S.K.T. acknowledges Ministry of Human Resource Develop-ment (MHRD) India for Ph.D. fellowship. We thank the Departmentof Biotechnology (DBT), India, Department of Science and Technol-ogy (DST), India, for financial support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2011.04.052.

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