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J. Biol. Chem., Vol. 282, Issue 27, 19928-19937, July 6, 2007
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From the
Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland, the Paul Scherrer Institute, Biomolecular Research, 5232 Villigen, Switzerland, and the ¶Max-Planck-Institut für Molekulare Physiologie, Strukturelle Biologie, 44227 Dortmund, Germany
Received for publication, January 19, 1007 , and in revised form, April 4, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The guanine nucleotide-binding domain (G domain) is about 20 kDa in size and structurally conserved. The G domain adopts an 
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fold, typical for many nucleotide-binding proteins, formed by six central -strands surrounded by five -helices. At the sequence level, the G domain contains five characteristic sequence motifs, termed G1-G5, involved in nucleotide binding and hydrolysis (2). The G1/Walker A motif (GXXXXGK(S/T)), also referred to as P-loop (4), helps to position the triphosphate moiety of the bound nucleotide. The G2 (X(T/S)X) and G3/Walker B (hhhDXXG) motifs are involved in the coordination of a Mg2+ ion that is required for nucleotide binding and hydrolysis. Specificity in nucleotide binding is conferred by the G4 motif, which has a (N/T)KXD signature in guanine nucleotide binding P-loop NTPases. The G5 motif ((T/G)(C/S)A) supports guanine base recognition (2).
The conformational switch between the GDP- and GTP-bound forms of P-loop GTPases manifests itself in two peptide segments defined as switch I and switch II. The switch I region, a loop preceding strand 2, has also been termed the effector loop because it exerts downstream functions in many GTPases by binding to effector molecules. The switch I and II regions interact with the 
-phosphate of the bound GTP, resulting in a protein conformation that is highly responsive to GTP hydrolysis and loss of the -phosphate. Upon GTP hydrolysis, the molecule undergoes a conformational change, and the switch domains move into a different position and conformation, a mechanism that has been compared with the relaxation of a loaded spring (3).
P-loop GTPases can be classified into TRAFAC (translation factor-related) and SIMIBI (signal recognition particle, MinD, and BioD) NTPases (5). Based on conserved sequence motifs and structural features, these two classes are further split into more than 20 distinct families and about 60 subfamilies (5).
Bacteria contain fewer GTPases than eukaryotes, with less diverse functions (6-8). Apart from those involved in translation, bacteria possess very few GTPases, and archaea have even fewer. Bacteria with small genomes (e.g. Mycoplasma genitalium) contain as few as 11 GTPases (6, 9). Strikingly, most of these GTPases are universally conserved and can be assigned to four main ancestral groups: elongation factors (EF-G, EF-Tu, and IF2), protein secretion factors (FtsY and Ffh), Era-related GTPases (Era, EngA, and ThdF/TrmE), and Obg-related proteins (Obg and YchF) (5, 6). Mutational analyses in many organisms have shown that most of these GTPases are essential (7, 10).
YchF is one of the highly conserved GTPases (Escherichia coli to Homo sapiens: 45% identity, 62% similarity) belonging to the Obg-related GTPase family. The Obg protein family is thought to comprise five ancient subfamilies, namely Obg/CgtA, YchF/YyaF, Drg/Rbg, Nog1, and Ygr210 (5). Functionally, the Obg-like GTPases are poorly characterized. The best-studied member is the essential Obg/CgtA (SpoOB-associated GTP-binding protein) from Bacillus subtilis (11). Obg and its homologues have been implicated to function in cellular processes as diverse as sporulation, stress response, control of DNA replication, and ribosome assembly (for review see 12).
In contrast to Obg, YchF/YyaF has been hardly investigated, and functional information on YchF homologues is scarce. In Brucella melitensis, the YchF homologue DugA has been suggested to be involved in iron metabolism and may be a regulator of the Ton system (13). Structural analysis of yeast and bacterial YchF revealed a modular organization formed by three domains, a central G domain, flanked by a coiled-coil domain and a TGS (ThrRS, GTPase, SpoT) domain of unknown function (14).
One anomaly in YchF and its homologues is the unusual G4 motif, which strongly diverges from the (N/T)KXD consensus, suggesting that guanine nucleotide binding specificity of YchF homologues might be either governed by other means or altered. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP.5 Our structural data help to explain the altered nucleotide specificity of YchF homologues. Biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPase.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cloning and Site-directed Mutagenesis—For hOLA1 cloning, the human gene PTD004 coding region was amplified by PCR using cDNA from HeLa cells. The PCR product was digested with NcoI and BamHI and inserted into the pQE60 protein expression vector (Qiagen) to allow for the expression of a C-terminally hexahistidine-tagged protein. The coding regions of S. cerevisiae Ybr025c (yOla1p) was amplified by PCR from yeast genomic DNA. The PCR product was digested with NcoI/BamHI and inserted into pQE60.
Point mutations were generated using the QuikChange® site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. DNA oligonucleotides used for PCRs and mutagenesis are listed in supplemental Table S1.
Protein Expression and Purification—Expression and purification of B. subtilis Obg and Hemophilus influenzae YchF was performed as described elsewhere (14, 16). Expression of all other proteins was done in E. coli BLR (pRep4). Protein expression was induced by the addition of 1 mM isopropyl -D-thiogalactopyranoside and performed overnight at 19 °C. The cells were lysed by cell cracking (EmulsiFlex-5C, Avestin) in 50 mM Tris, pH 7.6, 700 mM NaCl, 5 mM MgCl2, 2 mM 2-mercaptoethanol, 5% glycerol (w/v). The bacterial cell lysate was cleared by ultracentrifugation (90 min, 48,000 rpm, Ti70; Beckman) and was passed over nickel-nitrilotriacetic acid-agarose (Qiagen). After intense washing with lysis buffer, bound protein was subjected to buffer exchange (hOLA1 elution buffer: 50 mM Tris, pH 7.0, 100 mM NaCl, 5 mM MgCl2, 2 mM 2-mercaptoethanol, 5% glycerol (w/v); yOla1 elution buffer: 50 mM Tris, pH 6.5, 100 mM NaCl, 5 mM MgCl2, 2 mM 2-mercaptoethanol, 5% glycerol (w/v)) and then eluted with 400 mM imidazole in the corresponding elution buffer. The eluted protein was subsequently bound to a Hi Trap SP Sepharose HP column (GE Healthcare) and eluted in a gradient (buffer A: 50 mM Tris, 50 mM NaCl, 5 mM MgCl2, 5% glycerol (w/v); buffer B: 50 mM Tris, 1 M NaCl, 5 mM MgCl2, 5% glycerol (w/v) at the respective pH). Peak fractions were pooled and concentrated by Centricon centrifugation (Milian) prior to gel filtration on a Superdex 200 10/30 column (GE Healthcare) in 50 mM Tris, pH 7.6, 100 mM NaCl, 5 mM MgCl2. Peak fraction were pooled and supplemented with 2 mM dithiothreitol and 250 mM sucrose. Purified proteins were 98-99% pure and 95-98% nucleotide-free, as analyzed by SDS-PAGE and HPLC analysis, respectively.
Fluorescence Spectrometry—Nucleotide binding of 2'/3'-O-(N-methylanthraniloyl)-labeled nucleotides (mant-nucleotides; Jena Bioscience) was examined by fluorescence spectrometry (Cary Eclipse, Varian). All of the assays were performed at 25 °C, and mant-nucleotide fluorescence was monitored at an excitation wavelength of 355 nm (slit width, 5 nm) and an emission wavelength of 448 nm (slit width, 10 nm). The experiments were carried out in 50 mM Tris, pH 7.6, 100 mM NaCl, 5 mM MgCl2. hOLA1 was titrated to a 0.5 µM mant-nucleotide solution up to a final concentration of 15 µM. Alternatively, nucleotide binding was followed by polarization measurement with a Fluoromax 2 spectrofluorometer (Spex Industries). Mant-nucleotides were used at a concentration of 0.2 µM.
Isothermal Titration Calorimetry—Nucleotide binding affinity was determined by isothermal titration calorimeter (MCS-ITC, MicroCal, Inc.). Calorimetric experiments were performed at 25 °C in 50 mM Tris, pH 7.6, 200 mM NaCl, 5 mM MgCl2, 2 mM dithiothreitol. The concentration of protein in the sample cell was 58 µM, and the titrated nucleotide were used at 600 µM. The data were analyzed using the manufacturer's software. Curve fitting were done according to single ligand binding equations.
Nucleotide Hydrolysis Assay—To quantify nucleotide hydrolysis, 5 µM protein was incubated with an excess of either ATP or GTP (125 µM) in 50 mM Tris, pH 7.6, 200 mM NaCl, 5 mM MgCl2 at 25 °C. At various time points, nucleotide diphosphate production was analyzed by HPLC (Shimadzu). The nucleotides were separated on a hydrophobic C18-column (Macherey-Nagel) with 50 mM potassium-phosphate, pH 6.5, 10 mM tetrabutylammonium bromide, 5% acetonitrile as polar, mobile phase. Protein was denatured on a C-18 precolumn (Bischoff-Chromatography). For Fig. 7B, the nucleotides were separated by anion exchange chromatography on a Nucleosil 4000-7 PEI column (Macherey-Nagel) using a linear salt gradient (buffer A: 10 mM Tris/HCl, pH 8.5; buffer B: 10 mM Tris/HCl, pH 8.5, 1.5 M NaCl).
Alternatively, nucleotide hydrolysis was quantified using [-32P]ATP/GTP. In brief, the proteins were incubated with [-32P]ATP/GTP (50 µM, 0.05 µCi), and nucleotide hydrolysis reactions were stopped by the addition of 1 ml of a charcoal suspension. After centrifugation at 10,000 x g, release of [32P]phosphate was determined by scintillation counting of the supernatant. Competition was done with 50 µM of unlabeled nucleotides.
Crystallization and Data Collection—hOLA1 was incubated with 0.5 mg/ml trypsin to remove flexible parts of the protein and subsequently purified by gel filtration. Rod-like crystals of hOLA1 approximately 0.4 x 0.1 x 0.1 mm in size were obtained at 4 °C by vapor diffusion using the hanging drop method and 12% polyethylene glycol 3350, 50 mM Tris/HCl, pH 8, 10 mM MgCl2 as crystallization buffer. The nonhydrolyzable ATP analogue AMPPCP was added to the protein drop to a final concentration of 1 mM. The crystals were soaked with 30% glycerol as cryo-protectant, mounted using a nylon-fiber loop (Hampton Research), and flash-cooled to 100 K in a nitrogen stream. The data were collected at Beamline X06SA (SLS, Villigen) as consecutive series of 0.5° rotation images on a MARMOSAIC 225 detector. The data sets were indexed and integrated with XDS (17), scaled with XSCALE, and converted to CCP4, XPLOR, or SHELX format with XDSCONV. Data set statistics are given in Table 1.
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Superpositions—Superpositions were performed with the SSM superpose function within Coot. Briefly, hOLA1 full protein or the individual domains (G domain: residues 16-44, 58-65, 87-95, 107-126, and 198-305; coiled-coil: residues 139-171 and 180-195; TGS domain: residues 66-87 and 306-388) were superposed on full-length Thermus thermophilus YchF (Protein Data Bank code 2DBY), Schizosaccharomyces pombe YchF (Protein Data Bank code 1NI3), H. influenzae YchF (Protein Data Bank code 1JAL), and B. subtilis Obg (Protein Data Bank code 1LNZ). The quality of the superposition was judged on the sequence and optical alignment. Core root mean square deviation (RMSD) values are in Angstroms.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In general, Obg-related proteins share two conserved sequence features, namely an YXF(T/C)TXXXXXG segment in the switch I region and a glycine-rich motif in the switch II region (GAXXGXGXGXXX(I/L/V)). A distinguishing feature of the Ola1/YchF subfamily is a noncanonical G4 motif, distinct from the (N/T)KXD consensus (Fig. 1B). The G4 motif in the Ola1/YchF subfamily is as variable as NVNE in bacteria (YchF), NMSE in yeast (Ybr025cp/yOla1p), and NLSE in human (hOLA1). Both, the conserved lysine and aspartate are replaced. In contrast, the G1, G2, G3, and G5 motifs match the signature found in other P-loop GTPases (Fig. 1B).
The variation of the G4 motif may lead to an altered nucleotide binding specificity. Indeed, when we analyzed nucleotide binding of recombinant, highly purified hOLA1 using fluorescent, mant-labeled nucleotides by polarization analysis, we noticed that hOLA1 bound ATP. In contrast, we hardly observed GTP binding at a micromolar concentration of hOLA1 (Fig. 2A). Even binding of a nonhydrolyzable GTP analogue such as mant-GMPPNP was not saturable as shown by the linear dependence on hOLA1 concentration and failed to reach the level of ATP binding (see Fig. 6B). Further evidence that ATP is the preferred nucleotide-binding partner of hOLA1 stems from the observation that only ATP but not GTP could efficiently displace mant-ATP from hOLA1 (Fig. 2B). Together, this analysis shows that purified hOLA1 is an ATP-binding protein rather than a GTP-binding protein.
Isothermal titration calorimetry was used to determine the relative binding affinities for ATP and GTP. The dissociation constant (KD) for ATP was calculated to be 8 µM (Fig. 2C), whereas the KD for GTP could not be determined exactly because of its low affinity for hOLA1 (data not shown). Curiously, when the G4 motif of hOLA1 was reverted to the G4 consensus NKXD by site-directed mutagenesis, the hOLA1-NKXD mutant retained ATP-binding specificity (Fig. 2D), suggesting that the overall fold of the nucleotide-binding pocket in hOLA1 might define nucleotide specificity (see below).
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To obtain structural insight into ATP binding of hOLA1, we determined the crystal structure of hOLA1 complexed with AMPPCP, a nonhydrolyzable ATP analogue, at a resolution of 2.7 Å. The protein crystallized in space group C2221 (No. 20) with one monomer in the asymmetric unit and no obvious formation of higher oligomeric states in the crystal packing, consistent with the observation that the protein is a monomer in solution (static light scattering; data not shown). We could build 327 of 396 amino acids into the electron density map. Missing are the N-terminal residues 1-15, the switch I and switch II regions with residues 45-57 and 96-106, respectively, some disordered loop regions with residues 127-138, 172-179, 196-197, and the C-terminal residues 389-396. The model was completed by building AMPPCP and 26 water molecules into the electron density maps. The final model has a crystallographic R factor of 0.23 and a free R factor of 0.29 with very good stereochemistry (Table 1).
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atoms. The C atoms of the individual domains can be superimposed with RMSD values of 1.8 Å (G domain), 2.2 Å (coiled-coil domain), and 1.1 Å (TGS domain). Differences are more pronounced between hOLA1 and the GDP bound T. thermophilus YchF (Protein Data Bank code 2DBY, overall RMSD value 2.3 Å), the largest changes being observed for the coiled-coil domain (RMSD 2.5 Å), as well as helices 8b and 10 (for secondary structure assignment; see supplemental Fig. S3). The coiled-coil domain is tilted toward helix 7 in the latter structure, making it more compact. Helices 8b and 10, but not the coiled-coil domain, show similar changes in the superposition of the yeast and bacterial YchF structures, indicating a general variability in these structural elements.
The cofactor AMPPCP is bound in-between the P-loop (residues 28-36), helix 1, the adjacent helix 9, and the short loop following strand 6, which carries the unique Ola1 G4 motif N230LSE (Fig. 5A). The triphosphate moiety appears tightly bound through a network of H-bonds of main and side chain donors to the phosphate oxygens (Fig. 5B). The conserved interaction of the P-loop Lys35 with the -phosphates (via main chain NH) and -phosphates (via NH
) is also observed in hOLA1. We observe electron density at the potential binding site of the essential magnesium between Ser36 in the G1 P-loop, Thr55 in the G2-loop (switch I region), and the and -phosphates, but the resolution is too low to identify the ion or its position unambiguously.
The cofactor triphosphate moiety forms hydrogen bonds to residues from the P-loop, whereas its sugar moiety has no interaction with hOLA1, and its adenine base has only a few weak interactions with hOLA1. N-7 of the pyrimidine ring in the adenine base can form a hydrogen bond with NH
2 of Asn230 (distance 3.3 Å). The N-6 of the base can engage into a weak potential hydrogen bond to the main chain carbonyl oxygen of Leu231 with an N-O distance of 2.7 Å but a suboptimal C-O-N-angle of 103°. An interaction between the side chain carboxylate group of Glu233 and the nucleotide as proposed for GTP bound to YchF (14) appears not to be possible, because Glu233 is too far away.
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-strand conformation in hOLA1, it shows a zigzag-like conformation in Ras (31). Consistently, Ras Lys117 can stack with the cofactor, and Asp119 interacts with the N-1 and N-2 group of the guanine. In hOLA1, the individual positions of the G4 motif are shifted such that only position 1 (Asn230) matches with position 1 of Ras (Asn116), whereas position 3 of hOLA1 (Ser232) matches with position 4 of Ras (Asp119). The Glu233 of the hOLA1 G4 motif lies at the beginning of a small helix 8a preceding helix 8b, and intramolecular H-bonds force the entire motif into an extended conformation. This places Glu233 too far away to interact with the nucleotide. Apparently, the intramolecular forces that define the position of helices 8a and 8b do not allow the hOLA1 G4 motif to adopt a Ras-like conformation, explaining why the ATP specificity of hOLA1 could not be switched to GTP via mutations of the G4 motif to NKXD (Fig. 2D). To validate the role of key amino acids in the nucleotide-binding pocket of hOLA1, we mutated selected residues and tested these mutants for their ability to bind to mant-labeled ATP (Fig. 6A). As expected from the structural analysis, mutation of Asn230 to alanine (N230A) resulted in loss of ATP binding without perturbing solubility or other biochemical characteristics of the protein. Additionally, we mutated the conserved Phe127, which had been proposed to contribute to nucleotide binding in H. influenzae YchF through stacking of the aromatic side chain with the base (14). Indeed, when we replaced Phe127 with Ala (F127A), the mutant protein no longer bound mant-labeled ATP. Like wild-type hOLA1, both mutants also failed to bind GMPPNP or GTP (Fig. 6B and data not shown). This supports that Asn230 and Phe127 are essential for ATP binding. It seemed plausible that the conserved Phe127, that we could not see in the electron density map abolished proper conformation of the G4 motif and, together with the unconventional sequence in G4, would prevent efficient binding of GTP. However, combining the F127A and hOLA1-NKXD mutations did not result in a version of hOLA1 that could bind GTP (Fig. 6B), indicating that the overall fold of the nucleotide-binding pocket in hOLA1 must in addition be determined by other parameters.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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It is assumed that P-loop ATPases and GTPases have a common P-loop-containing ancestor. Several ATPases have been derived secondarily form the GTPase superclass through loss of GTPase specificity or activity (5). The kinesinmyosin superfamily of motor ATPases, for instance, may have evolved from the TRAFAC class by loss of the NKXD G4 motif, accompanied by a the gain of ATPase activity. Another example is the subfamily of MinD-like ATPases, which derived within the SIMIBI class of the GTPase superclass (5). The Ola1/YchF subfamily of the Obg family of GTPases, however, presents a unique exception of a single protein subfamily, which shows altered nucleotide specificity within a distinct protein family of GTPases, whereas in the aforementioned examples whole protein families of distinct nucleotide specificity evolved. This implies that in the case of the Ola1/YchF subfamily the change in nucleotide specificity occurred relatively late in the evolution of the Obg family.
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The biochemical characterization of the ATPase activity of hOLA1 was supported by structural analysis of hOLA1 complexed with AMPPCP. The interactions between the bound adenine nucleotide and hOLA1 mainly involve the phosphate groups of the bound AMPPCP. The -phosphate contacts the main chain amide of Thr37, the -phosphate the main chain amides of Ser36 and Val33, and the -phosphate forms a hydrogen bond to the main chain amide of Asn32 in hOLA1. In our structure, we cannot observe any contacts of the ribose to the protein.
The specificity of nucleotide interactions must be determined by contacts of the protein with the adenine base. The (unprotonated) N-7 of the adenine base forms a hydrogen bond with NH2ofAsn230. The N-6 group of the base might engage into a hydrogen bond with the main chain CO of Leu231, which is part of the unconventional G4 motif of hOLA1. The distance of 2.7 Å falls well within acceptable H-bond range, although the geometry is not optimal in the refined model. Therefore, we cannot exclude an energetically more favorable orientation of the peptide carbonyl group. Our biochemical data, however, support an important contribution of Asn230 to nucleotide binding, because its mutation to alanine abolished nucleotide binding. Another contact from the protein contributing to base recognition might be established by Phe127. Mutating this residue to Ala diminishes ATP binding drastically, indicating an important involvement of this residue in nucleotide binding. The electron density for Phe127 is too poorly defined for the residue to be modeled. Phe127 may contribute to the fixation of the nucleotide via stacking on the base, as proposed for the H. influenzae YchF case (14).
So far, there is no published structure of an Ola1-like protein bound to a nucleotide-triphosphate. Structures of YchF from H. influenzae (14) and S. pombe (Protein Data Bank code 1NI3) have been solved in the nucleotide-free form. The fact that Teplyakov et al. (14) were not able to obtain a YchF-GTP cocrystal structure by soaking or cocrystallization supports our results on the binding preference of hOLA1 for ATP and suggests that this specificity extends to other members of the Ola1/YchF subfamily. Very recently, a structure of YchF from T. thermophilus bound to GDP has been published in the data base (Protein Data Bank code 2DBY). However, in that structure, there is no interpretable electron density for the ribose and the base. Therefore, it is not clear how the base is recognized in this case. Our biochemical data clearly show that ATP binding to Ola1-like proteins is favored over GTP binding, but that GTP association can be forced to occur at high nucleotide concentration in vitro.
How can the preference of hOLA1 for ATP be explained? The nucleotide binding mode deduced from our structure suggests that the relative specificity of hOLA1 for adenine and discrimination against guanine binding is based on the interaction between the adenine N-6 group and Leu231 main chain CO in G4. In guanine, with the carbonyl oxygen O-6 present at the 6-position, this hydrogen bond binding mode is not possible. Instead, repulsive forces between the O-6 and Leu231 CO presumably disfavor guanine binding (Fig. 5C).
The ATP binding specificity of hOLA1 appears even more striking when compared with the founding member of the family, B. subtilis Obg bound to ppGpp (16). The 5' phosphates of both nucleotides (AMPPCP and ppGpp) superimpose very closely, with equivalent H-bond patterns (supplemental Fig. S4). Strikingly, both the ribose and base moieties are tilted by about 40° with respect to one another. The different angle of the adenine base relative to the phosphates in AMPPCP allows for the adenine N-6-Leu231O H-bond in the hOLA1 case, whereas a different hydrogen bond is formed between the ppGpp and Obg (N-1-Asp285O2 and N-2-Asp285O1). This latter, bidentate interaction represents in fact the only clear discrimination between adenine and guanine base, because the H-bond between the ppGpp O-6 and Ser310O could also be formed with an exocyclic N-6 of an adenine in a similar position.
The G4 motifs of the GTPase Ras (116NKCD) and the ATPase hOLA1 (230NLSE) show different conformations (Fig. 5B). Although the G4 motif in Ras adopts a zigzag-like conformation (31), the G4 motif in hOLA1 possesses an extended -strand conformation. In the G4 motif of hOLA1, Ser232 and Glu233 cannot interact with the nucleotide because of the altered conformation of the G4 loop. Our attempts to convert hOLA1 back into a GTPase by conversion of the G4 motif to NKXD did not result in a version of hOLA1 that could bind GTP but retained ATP specificity (Fig. 2D). This indicates that the overall fold of the nucleotide-binding pocket in hOLA1 is determined by additional parameters.
In Ras-like GTPases, a Gln residue is involved in (GAP-induced) GTP hydrolysis. This conserved Gln residue is replaced by a hydrophobic amino acid in all Obg family members (e.g. Leu96 in hOLA1), a substitution that inactivates Ras-like GTPases (32). This indicates a different NTP hydrolysis mechanism used by Obg-like NTPases. P-loop NTPases undergo conformational changes upon NTP hydrolysis. The structural differences between AMPPCP-bound hOLA1 and nucleotide-free YchF are minimal, and it remains to be seen which structural differences might exist between ATP and ADP-bound hOLA1.
Ola1-like proteins display a low intrinsic nucleotide hydrolysis and a high nucleotide dissociation rate, as observed for Obg, the founding member of the Obg family. It remains to be seen whether these features are important for hOLA1 function in vivo or whether yet to be identified binding partners of Ola1-like proteins influence their nucleotide binding and hydrolysis properties in the cellular context. This awaits the identification of potential binding partners and their functional characterization.
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FOOTNOTES |
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* This work was supported by an intramural ETH grant (to R. K.-E. and U. K.) and by funding through the Swiss National Science Foundation (to U. K. and C. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and supplemental Figs. S1-S4.
1 These authors contributed equally to this work.
2 Present address: Bayer HealthCare AG, D-42096 Wuppertal, Germany.
3 To whom correspondence may be addressed: Institute of Biochemistry, HPM F11.1, Schafmattstr.18, ETH Zurich, 8093 Zurich, Switzerland. Tel.: 41-44-632-3013; Fax: 41-44-632-1591; E-mail: ulrike.kutay{at}bc.biol.ethz.ch.
4 To whom correspondence may be addressed: Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland. Tel.: 41-56-310-4723; Fax: 41-56-310-5288; E-mail: christian.kambach{at}psi.ch.
5 The abbreviations used are: AMPPCP, adenosine 5'-(,-methylenetriphosphate); HPLC, high pressure liquid chromatography; RMSD, root mean square deviation; GMPPNP; guanosine 5'-(,-imido) triphosphate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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H, enthalpy change; 
