Biochemical properties of the Vibrio harveyi CgtAV GTPase

BBRC Biochemical and Biophysical Research Communications 339 (2006) 1165–1170 www.elsevier.com/locate/ybbrc

Biochemical properties of the Vibrio harveyi CgtAV GTPase A.E. Sikora a

a,b

, K. Datta a, J.R. Maddock

a,*

Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109, USA b Department of Molecular Biology, University of Gdansk, Kładki 24, 80-822 Gdansk, Poland Received 28 October 2005 Available online 5 December 2005

Abstract Bacteria encode a number of relatively poorly characterized GTPases, including the essential, ribosome-associated Obg/CgtA proteins. In contrast to Ras-like proteins, it appears that the Obg/CgtA proteins bind guanine nucleotides with modest affinity and hydrolyze GTP relatively slowly. We show here that the Vibrio harveyi CgtAV exchanges guanine nucleotides rapidly and has a modest affinity for nucleotides, suggesting that these features are a universal property of the Obg/CgtA family. Interestingly, CgtAV possesses a significantly more rapid GTP hydrolysis rate than is typical of other family members, perhaps reflecting the diversity and specificity of bacterial ecological niches.
2005 Elsevier Inc. All rights reserved. Keywords: GTPase; Obg; CgtA; GTP hydrolysis; Guanine nucleotide exchange

The Obg/CgtA proteins form a distinct group of monomeric GTPases that are highly conserved from bacteria to humans. These GTPases are also ribosome associated and likely play a role in the assembly of the large ribosomal subunit. Direct binding to 50S ribosomal subunits has been demonstrated for CgtA in Caulobacter crescentus [1], Escherichia coli [2,3], Vibrio harveyi [4], as well as for the Saccharomyces cerevisiae proteins Mtg2p [5] and Nog1p [6,7]. In Bacillus subtilis, Obg cofractionates with ribosomes [8] and binds to ribosomal protein L13, as detected by affinity blot assay [9]. Either depletion or mutation of all Obg/CgtA proteins examined thus far results in a ribosome assembly defect [1,5,10]. Moreover, overexpression of the Escherichia coli protein CgtAE (also called YhbZ and ObgE) suppresses both the cold sensitivity and ribosome assembly defects of an rrmJ mutant [11]. RrmJ is a methyltransferase that modifies the 23S rRNA [12,13] at a late step in the maturation process of the 50S subunit. Furthermore, it has recently been shown that processing of the 16S rRNA is defective in a cgtAE mutant [3]. The terminal pro-

*

Corresponding author. Fax: +1 734 647 0884. E-mail address: [email protected] (J.R. Maddock).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.129

cessing of the 16S rRNA occurs after coupling of the 30S and 50S subunits, and 50S assembly mutants have been shown to result in the accumulation of unprocessed 16S rRNA [14,15]. Finally, the E. coli CgtAE protein co-purifies with a 50S biogenesis protein, CsdA [2,3]. Taken together, these data strongly suggest that the Obg/CgtA proteins are involved in biogenesis of the 50S ribosomal particle. In addition to a role in ribosome assembly, the Obg/ CgtA proteins are also involved in stress response. Depletion of B. subtilis Obg results in a sporulation defect [16]. In B. subtilis, Obg interacts with RsbW, RsbT, and RsbX, three proteins involved in the activation of rB, a ribosomeassociated stress response factor [9,17]. Curiously, the B. subtilis Obg protein co-crystallized with the SpoT/RelA product, ppGpp [18], raising the possibility that this nucleotide may play a role in the control of the Obg/CgtA proteins. Consistent with this possibility, the E. coli CgtAE protein interacts with the ppGpp synthetase/hydrolase protein SpoT [2]. The biochemical features of the Obg/CgtA proteins are distinct from those of eukaryotic Ras-like proteins. All Obg/CgtA proteins examined thus far have modest affinity for guanine nucleotides, rapidly exchange guanine nucleotides, and display relatively slow GTP hydrolysis rates

A.E. Sikora et al. / Biochemical and Biophysical Research Communications 339 (2006) 1165–1170

Materials and methods Bacterial strains, plasmids, and growth conditions. Escherichia coli cells were grown at 37 C (unless otherwise indicated) in Luria–Bertani media (LB; 10 g tryptone, 5 g yeast extract, and 10 g NaCl/l) or on LB agar (1.5% agar) containing 100 lg/ml ampicillin or 30 lg/ml kanamycin. Cloning and plasmid amplification were performed in E. coli DH5a [29]. Expression and purification of His-tagged CgtAV. Full-length cgtAV was PCR amplified using primers 5 0 -GGCATATGAAGTTCGTAGATGA and 5 0 -ATGGATCCGCAAATCACATCGTCT from V. harveyi BB7 [30]. An N-terminally His-tagged CgtAV was created by cloning the 1.3 kb Nde1 to BamHI fragment into pET28a(+) (Novagen) to create pAES2. His-CgtAV was expressed from pAES2 in E. coli BL21(DE3) by induction with 0.5 mM IPTG for 3 h at 30 C and purified as previously described [2]. Approximately 50% of the His-CgtAV protein, hereafter called CgtAV, was in the supernatant and the final yield was 30 mg/L. UV cross-linking. UV cross-linking reactions were performed as described previously [19]. For competition assays, purified CgtAV (20 lM) was incubated with 10 lCi [a-32P]GTP [3000 Ci/mmol (NEN Life Science Product)] in 50 ll binding buffer B [50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2 mM dithiothreitol, 10 lM ATP, 1 mM EDTA, and 10 % (wt/vol) glycerol] supplemented with 5 mM Mg2+, in the presence or absence of 40 lM nonradioactive GTP, GDP, GMP, ATP, CTP or UTP. The Mg2+ concentration dependence of CgtAV–[a-32P]GTP complex formation was investigated by incubation of purified CgtAV (12 lM) with 0.5 lM [a-32P]GTP [3000 Ci/mmol (NEN Life Science Product)] in binding buffer B (50 ll) supplemented with 0, 0.1, 0.5, 1, 3, 5 or 12 mM Mg2+. Fluorescence measurements. Fluorescence measurements were performed as described [10,19]. To determine the guanine nucleotide binding ability of CgtAV, 4 lM purified protein was prebound to 0.3 lM mantGTP or mant-GDP in binding buffer B in the presence or absence of 5 mM Mg2+, as indicated. The Mg2+ dependence of mant-nucleotides–CgtAV complex formation was determined by monitoring the mant-GTP and mant-GDP fluorescence of 2.3 lM purified protein bound to 0.3 mantGTP or -GDP in buffer B (without EDTA) supplemented with 0–50 mM Mg2+. To obtain dissociation rate constants (kd), CgtAV (11.5 lM) was prebound to 1 lM mant-nucleotide in binding buffer containing 5 mM or

0 mM Mg2+ (for mant-GTP and mant-GDP, respectively). Two hundred micro molars of competitor GDP or GTP was rapidly added using a stopflow fluorometer, the decrease in fluorescence was measured, and the data were fitted to a single-phase exponential decay exponential equation using the GraphPad Prism Software version 3.0 for Windows (San Diego, California). The dissociation rate constants for both mant-nucleotides were obtained by averaging rates from 10 experiments. To ascertain the rate of mant-GTP hydrolysis by CgtAV, 16 lM of purified protein was prebound to 0.3 lM mant-GTP in binding buffer supplemented with 5 mM Mg2+. The GTPase activity of CgtAV was determined by monitoring the decrease in fluorescence of the CgtAV– mant-GTP complexes over 3 h at 1 min intervals. Data were fitted to single exponential decay equation using GraphPad Prism software. The single turnover rate constant and the half-life of hydrolysis were obtained by averaging data from four trials.

Results CgtAV binding to GTP but not GDP requires magnesium The Obg/CgtA proteins examined thus far have modest affinities for guanine nucleotides, rapid nucleotide exchange rate constants, and relatively slow GTPase activities [2,11,18–20]. To determine whether this was also the case for CgtAV, we expressed in E. coli and purified both N- and C-terminally His-tagged CgtAV proteins. The majority (>60%) of the C-terminally tagged CgtAV protein, even when isolated from E. coli BL21(DE3) cells grown at 16 C, was insoluble (data not shown). Moreover, the soluble CgtAV-His bound to mGDP weakly and had no detectable affinity for mGTP, as detected by fluorometry (data not shown). In contrast, the N-terminally His-tagged CgtAV was soluble. Like the E. coli [2] and C. crescentus CgtAC [19] proteins, CgtAV migrated more slowly by SDS–PAGE than predicted (
47 kDa vs.
43 kDa, data not shown). The ability of purified His-CgtAV to bind to radiolabeled GTP was assayed directly by UV crosslinking (Fig. 1A). We assayed the ability of GTP, GDP, GMP, ATP, CTP, and UTP to compete for binding of His-CgtAV to [a-32P]GTP. Both GTP and GDP were potent competitors, whereas excess GMP, ATP, CTP or UTP had little effect on the binding of CgtAV to [a-32P]GTP (Fig. 1A). Thus, CgtAV is a specific GTP and GDP-binding protein.

A Competitor

B Mg2+ (mM)

0

+G TP +G DP +G MP +A TP +C TP +U TP

[2,11,18–20]. Similar biochemical properties are also found in other bacterial GTPases predicted to play roles in ribosome function such as the E. coli Era [21], EngA (also called Der) [22], YjeQ (also called RsgA) [23], and YihA (also called EngB) [24] proteins. One possibility is that, unlike eukaryotic Ras proteins, some or all of these ribosome-associated bacterial GTPases are controlled by the cellular guanine nucleotide pools. Such a model is attractive, as it would provide a mechanism by which to couple ribosome assembly with cell cycle control. The role of the V. harveyi CgtAV protein is unknown. The literature on CgtAV is complicated due to a series of publications characterizing a strain, BB7X, which was reported to have a viable insertion mutation in cgtAV [25–28]. We have recently shown that the mutant phenotype in strain BB7X was not due to a transposon insertion in cgtAV and that cgtAV is an essential gene [4]. Here we report the biochemical characterization of the CgtAV protein. These studies show that the guanine nucleotide binding and exchange rate constants of CgtAV are similar to those of other Obg/CgtA proteins whereas the GTP hydrolysis rate is approximately 10-fold faster, perhaps reflecting differences in the lifestyle and environment of this marine bacterium.

no ne

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0.1

0.5

1

3

5

12

Fig. 1. CgtAV is a GDP and GTP binding protein. (A) The guanine nucleotide specificity of purified CgtAV in the presence of 5 mM Mg2+ was determined by addition of competing nucleotide (
600-fold), as indicated prior to UV crosslinking. (B) CgtAV–GTP complex formation requires Mg2+. The indicated levels of Mg2+ were added prior to UV crosslinking. Shown are autoradiograms of UV crosslinked CgtAV–[a-32P]GTP complexes separated by SDS–PAGE.

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The binding of GTP, but not GDP, is dependent on the presence of Mg2+ in both E. coli [2] and C. crescentus [19]. To determine whether this was also the case for CgtAV, we assayed the binding of [a-32P]GTP in the presence of various concentrations of Mg2+. In the absence of Mg2+, CgtAV did not bind to GTP, whereas strong binding was observed in the presence of 1–12 mM Mg2+ (Fig. 1B). The fluorescent guanine nucleotide analogs mant-GTP and mant-GDP are useful for examining the GTP hydrolysis and guanine nucleotide exchange of bacterial GTPbinding proteins [2,19,21,24,31–34]. Binding of CgtAV to mant-GTP and mant-GDP led to an increase in mant-nucleotide fluorescence (1.8- and 1.4-fold, respectively) at the excitation wavelength of 361 nm (Fig. 2A). We monitored the increase in fluorescence obtained upon binding to the mant-nucleotides to address whether CgtAV required Mg2+ for optimal binding to mant-nucleotides. The binding of CgtAV to mant-GTP was also Mg2+ dependent, with optimal binding, as indicated by maximal fluorescence, 2

Relative Fluorescence

A

1.5 1

Relative Fluorescence (% maximal)

occurring between 5 and 10 mM Mg2+ (Fig. 2B). In contrast, the binding to mant-GDP was relatively unaffected by the Mg2+ concentration. Thus, CgtAV requires Mg2+ for optimal binding to GTP but not GDP, a requirement similar to that observed for other Obg/CgtA proteins. The guanine nucleotide exchange rate of CgtAV is rapid We used the difference in fluorescence between bound and free mant-nucleotides to measure the guanine nucleotide exchange rates for mant-GTP and mant-GDP, as we have done previously [2,19,21,31,32]. CgtAV was prebound to mant-GTP in the presence of 5 mM Mg2+, or to mantGDP in the absence of Mg2+, until apparent saturation was achieved. Excess unlabeled nucleotide (GTP or GDP) was added and the decrease in fluorescence monitored (Figs. 3A and B). We obtained dissociation rate constants for mantGTP and mant-GDP of 0.23 ± 0.01 s1 and 0.3 ± 0.01 s1, respectively. Similar rates were obtained regardless of whether GDP or GTP was used as the competing nucleotide (data not shown). The guanine nucleotide dissociation rate constants for CgtAV are comparable to those seen for the CgtA proteins from E. coli [2] and C. crescentus [19]. Thus, it appears that all Obg/CgtA proteins display rapid guanine nucleotide exchange in vitro. GTP hydrolysis by CgtAV is faster than that seen in other Obg/CgtA proteins

0.5

310

B

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361 Excitation Wavelength (nm)

410

110 100 80

We also utilized mant-nucleotides to measure the single turnover hydrolysis rate of CgtAV. The decrease in fluorescence that accompanied the hydrolysis of CgtAV-bound mant-GTP to mant-GDP was monitored (Fig. 3C) and fitted to a single exponential curve with a first rate-order rate constant, kh, of 5.2 · 103 s1 or T1/2 of 2.21 ± 0.2 min. Thus, the intrinsic hydrolysis rate of CgtAV is approximately 10· faster than that of the E. coli [2,11] or C. crescentus [19] CgtA proteins. Discussion

60 40 20 0 0

0.1 0.5 1 Mg 2+ concentration (mM)

5 10

Fig. 2. Guanine nucleotide binding, Mg2+ dependence, and GTP hydrolysis using mant nucleotides. (A) Excitation spectra recorded at an emission wavelength of 446 nm in the presence of 5 mM Mg2+. The fluorescence intensity of the mant moiety in the absence of protein is identical whether mant is coupled to GDP or to GTP (represented by superimposed gray line). Upon addition of CgtAV, the fluorescence intensity of both mGDP (dashed line) and mGTP (solid line) increases. (B) CgtAV binds mGDP and mGTP with nucleotide-specific Mg2+ dependence. Binding of mGDP (triangles) and mGTP (squares) to CgtAV was assayed in the presence of various concentrations of Mg2+.

It is becoming clear that the Obg/CgtA proteins play similar, but slightly different, roles in various bacteria. It is likely that all bacterial Obg/CgtA proteins are associated with the large ribosomal subunit and are involved in late 50S ribosome assembly. Additionally, they all appear to play a role in stress response but the specific mechanisms by which this occurs may vary. Stress response in all bacteria is mediated, at least in part, through a SpoT-like ppGpp hydrolase/synthetase. A few bacteria, such as E. coli and V. harveyi, also express a related ppGpp synthetase, RelA. In addition to controlling ppGpp levels, some sporulating bacteria, such as B. subilis, also regulate an alternative sigma factor (rB) in response to stress. Interestingly, recent studies in B. subtilis show that RelA is also involved in regulation of rB [35]. Given that the Obg/CgtA proteins have been shown to interact with SpoT [2] in E. coli and with

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A

kd GDP = 0.36 ± 0.01 s-1

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800 750 700 650 600 550 0

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600

Relative Fluorescence

kd GTP = 0.23 ± 0.01 s-1 550

500

450

400 0

5

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Relative Fluorescence

C

500

475

450

425 0

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40

Time (min) Fig. 3. CgtAV displays rapid guanine nucleotide exchange and a relatively rapid GTP hydrolysis rate. Dissociation of (A) mant-GDP and (B) mantGTP. CgtAV–mant-nucleotide complexes (generated by prebinding mantGTP or mant-GDP with CgtAV) were rapidly mixed with excess GTP in a stopped-flow fluorometer, and the change in the relative fluorescence intensity over time was monitored. Representative profiles for the dissociation of guanine nucleotides are shown. The curve-fitted data are shown with a solid line. (C) Hydrolysis of mant-GTP was monitored by the decrease in fluorescence that accompanies the conversion of mantGTP to mant-GDP was recorded over time. Data were fitted to a single exponential decay equation. Shown are the data from a representative experiment.

proteins that regulate rB in B. subtilis [9], Obg/CgtA may be a key global stress response protein with slightly different partners depending on its ecological niche. How

ribosome association, ribosome assembly, and stress response control are coordinated is unknown and its understanding will require molecular details of Obg/CgtA protein function. Towards this end, it is critical to define the nature of the GTP/GDP (and perhaps ppGpp) cycle of the Obg/CgtA proteins. A common feature of these proteins, including the V. harveyi CgtAV protein, is their relatively low affinity for guanine nucleotides and rapid guanine nucleotide exchange rates [2,11,18–20] (Fig. 3), characteristics that differ from those of the small, eukaryotic Ras-like proteins. In C. crescentus, detailed studies of specific GTP binding pocket mutants have demonstrated that CgtAC mutations that affect Ras-like proteins in certain predictable ways affect CgtAC differently [10,32]. Therefore, we predict that the in vivo regulation of the Obg/CgtA proteins is different from that of the eukaryotic Ras proteins. One possible explanation for the Obg/CgtA vs. Ras kinetic differences is that association with the 50S ribosomal subunit in vivo inhibits rapid nucleotide exchange, allowing GTP hydrolysis to perform some cellular function, such as in ribosome assembly or controlling stress response. Another possibility is that guanine nucleotide occupancy would affect the ribosome association of the Obg/CgtA proteins as has been shown for B. sublitis Obg [8]. Curiously, the GTP hydrolysis rate of CgtAV is approximately 10-fold faster than that reported for any other Obg/CgtA protein. Whether this difference reflects a difference in the mechanism of CgtAV control or function is unknown. Interestingly, it appears that perturbations to the C-terminus of the Obg/CgtA proteins are deleterious to protein function. In this study, we found that a C-terminal CgtAVHis fusion protein was predominantly insoluble and bound GDP weakly, whereas an N-terminal His-tagged protein was soluble and active for binding guanine nucleotides. We have previously reported that the C-terminal domain of the C. crescentus CgtAC protein is critical for function [1]. Addition of a C-terminal 3HA-tag to CgtAC results in slow growth. Furthermore, whereas mutant CgtAC proteins CgtACT193A, or a C-terminal deletion of seven amino acids, although slightly impaired for guanine nucleotide binding, support growth, the addition of 3HA to the C-terminus of either of these mutant proteins abolishes viability. Moreover, the C-terminally tagged CgtAC proteins failed to associate with 50S particles when co-expressed with wild type protein, suggesting that the slow growth phenotype may be a consequence of poor ribosome association. Taken together, these data suggest a critical role for the C-terminus of the Obg/CgtA proteins [1]. These observations are particularly significant in light of a recent study of a E. coli CgtAE fusion protein generated from a transposon insertion that removes the last 9 amino acids and adds 68 amino acids encoded from one end of Tn5 [36]. This mutant results in severe sensitivity to DNA replication inhibitors, as well as replication forks vulnerable to breakage in a subpopulation of mutants. The authors conclude that CgtAE either directly or indirectly promotes replication fork

A.E. Sikora et al. / Biochemical and Biophysical Research Communications 339 (2006) 1165–1170

stability [36]. Furthermore, these phenotypes were similar to those of overexpression of a CgtAEP168V [36], a mutant protein that, like the CgtACP168V mutant protein [10], should be GTP-bound in vivo due to a reduced affinity for GDP. Clarifying the relationships among the role of the C-terminus, ribosome association, GTP-occupancy, and the effects on DNA replication is complicated but necessary for a better understanding of the role of Obg/CgtA proteins in the cell. Parallel studies in multiple, distantly related bacteria are critical for assigning both species-specific and global features to these universally conserved, essential GTPase proteins. Acknowledgments We are extremely grateful Susan Sullivan for careful critique of the manuscript. We are also grateful to Grzegorz We˛grzyn for insightful discussions during the course of this study. This work was supported by, a collaborative FIRCA grant (TW006001) from the National Institutes of Health and from NATO funds to JRM and Grzegorz We˛grzyn. References [1] B. Lin, D.A. Thayer, J.R. Maddock, The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit, J. Bacteriol. 186 (2004) 481–489. [2] P. Wout, K. Pu, S.M. Sullivan, V. Reese, S. Zhou, B. Lin, J.R. Maddock, The Escherichia coli GTPase, CgtAE, cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase, J. Bacteriol. 186 (2004) 5249–5257. [3] A. Sato, G. Kobayashi, H. Hayashi, H. Yoshida, A. Wada, M. Maeda, S. Hiraga, K. Takeyasu, C. Wada, The GTP binding protein Obg homolog ObgE is involved in ribosome maturation, Genes Cells 10 (2005) 393–408. [4] A. Sikora, R. Zielke, K. Datta, and J. Maddock, The Vibrio harveyi GTPase CgtAV is essential and associated with the 50S ribosomal subunit, J. Bacteriol. (2006) In press. [5] K. Datta, J.L. Fuentes, J.R. Maddock, The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2, Mol. Biol. Cell 16 (2005) 954–963. [6] B.C. Jensen, Q. Wang, C.T. Kifer, M. Parsons, The NOG1 GTPbinding protein is required for biogenesis of the 60S ribosomal subunit, J. Biol. Chem. 278 (2003) 32204–32211. [7] G. Kallstrom, J. Hedges, A. Johnson, The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively, Mol. Cell. Biol. 23 (2003) 4344–4355. [8] S. Zhang, W.G. Haldenwang, Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes, Biochem. Biophys. Res. Commun. 322 (2004) 565–569. [9] J.M. Scott, J. Ju, T. Mitchell, W.G. Haldenwang, The Bacillus subtilis GTP binding protein Obg and regulators of the rB stress response transcription factor cofractionate with ribosomes, J. Bacteriol. 182 (2000) 2771–2777. [10] K. Datta, J.M. Skidmore, K. Pu, J.R. Maddock, The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels, Mol. Microbiol. 54 (2004) 1379–1392. [11] J. Tan, U. Jakob, J.C. Bardwell, Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase, J. Bacteriol. 184 (2002) 2692–2698.

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