Crystal Structure of the YGR205w Protein From

PROTEINS: Structure, Function, and Bioinformatics 54:776 –783 (2004)

Crystal Structure of the YGR205w Protein From Saccharomyces cerevisiae: Close Structural Resemblance to E. coli Pantothenate Kinase Ines Li de La Sierra-Gallay,1 Bruno Collinet,2 Marc Graille,1 Sophie Quevillon-Cheruel,2 Dominique Liger,2 Philippe Minard,2 Karine Blondeau,3 Gilles Henckes,3 Robert Aufre`re,3 Nicolas Leulliot,2 Cong-Zhao Zhou,2 Isabelle Sorel,2 Jean-Luc Ferrer,4 Anne Poupon,1 Joe¨l Janin,1 and Herman van Tilbeurgh2* 1 Laboratoire d’Enzymologie et Biochimie Structurales (CNRS-UPR 9063), Baˆt. 34, 1 Av. de la Terrasse, 91198 Gif sur Yvette, France 2 Institut de Biochimie et de Biophysique Molećulaire et Cellulaire (CNRS-UMR 8619), Universite´ Paris-Sud, Baˆt. 430, 91405 Orsay, France 3 Institut de Geńe´tique et Microbiologie (CNRS-UMR 8621), Universite´ Paris-Sud, Baˆt. 360, 91405 Orsay, France 4 IBS J.-P. Ebel / LCCP 41, rue Jules Horowitz, 38027 Grenoble cedex 1, France

ABSTRACT The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three-dimensional crystal structure of Se-methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P-loop in a position that corresponds to the position of ␤-phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined. Proteins 2004;54:776 –783. ©

2004 Wiley-Liss, Inc.

Key words: structural genomics; kinase; P-loop; yeast; YGR205w; nucleotide; crystal structure; mononucleotide binding fold INTRODUCTION The sequencing of the Saccharomyces cerevisiae genome revealed the presence of about 6000 open reading frames.1 In order to discover the cellular function of the products of these genes, a number of large scale post-genomic studies have been conducted on this organism.2–5 However, a whole genome approach for the discovery of biochemical function remains beyond reach. Structural genomics initiatives endeavor to improve the gap between the rapidly growing world of sequences and that of protein 3D struc©

2004 WILEY-LISS, INC.

tures. This is being achieved through systematic highthroughput structure determination by crystallography and NMR. This explosive increase in experimental structures will allow to model more and larger protein families.6 –9 Structural genomics will also make an essential contribution to the functional characterization of all the proteins encoded by genomes.10 The structural genomics project conducted at the SouthParis campus area targets yeast proteins of unknown structure by either protein crystallography or NMR (“http:// genomics.eu.org/”).11 In a first phase, we concentrate our efforts on the structure determination of non-membrane proteins of size smaller than 500 amino acids for which no reliable structural model can be proposed. One of our targets, the YGR205w ORF codes for a 33 kDa protein (290 amino acids) of unknown function. A large-scale gene deletion study reported that the knock-out yeast strain for this gene is viable. It has clear sequence analogs in other yeast species and also in higher eukaryotes, but nothing is known on function or structure for any of these proteins. The only recognizable feature of the sequence is a [A/G]X(4)-G-K-[S/T] sequence motif, with X any amino acid, often involved in ATP/GTP-binding (P-loop motif).12 The presence of this loop may indicate that YGR205wp is a member of the large phosphotransferase family (EC 2.7). Many of these enzyme activities have no associated protein sequences yet. YGR205wp provides an interesting target for structural genomics, since its structure will add to our knowledge of the phosphotransferase family on the Abbreviations: MAD, multiple anomalous dispersion; ATP, adenosyl triphosphate; AMPPNP, 5⬘-adenylimido-diphosphate, Mant-ATP, Nmethylanthranyloyl-ATP, TMP, thymidylate monophosphate; SDS PAGE, sodium dodecyl sulphate poly acrylamide gel electrophoresis; ORF, open reading frame; r.m.s., root mean square. *Correspondence to: Herman van Tilbeurgh, Institut de Biochimie et de Biophysique Molećulaire et Cellulaire (CNRS-UMR 8619), Universite´ Paris-Sud, Baˆt. 430, 91405 Orsay, France. E-mail: [email protected] Received 3 April 2003; Accepted 26 July 2003

RESEMBLANCE OF S. CEREVISIAE YGR205W PROTEIN TO E. COLI PANTOTHENATE KINASE

one hand, and it offers an interesting case of structure function paradigm on the other. We present here the crystal structure of the YGR205w protein confirming that this protein belongs to the mononucleotide binding structural family. The structure has a typical ␣/␤ mononucleotide binding fold, revealing a strong structural relationship to Escherichia coli pantothenate kinase, but also to other small molecule and nucleotide kinases. The presence of well defined sulphate ions in the crystal structure and biochemical nucleotide binding experiments suggest that this protein may represent a small metabolite kinase. MATERIALS AND METHODS Cloning, Expression, and Purification The YGR205w gene was amplified by PCR using genomic DNA of S288C Saccharomyces cerevisiae strain as template and was cloned in the pCRT7/CT-TOPO vector from Invitrogen. An oligodeoxynucleotide (MWG-BIOTECH) coding for a 6-histidine tag was added at the 3⬘ end of the gene. The E. coli expression strain BL21(DE3)pLysS (Novagen) was transformed with the construct and grown in 2xYT medium (BIO101 Inc.) at 37°C up to an A600nm of 1. Expression was induced with 0.3 mM IPTG (Sigma) and the cells were grown for a further 4 hours at the same temperature. Cells were collected by centrifugation, suspended in 30 ml of a buffer containing 20 mM Tris-HCl, pH 8, 200 mM NaCl and 5 mM ␤-mercaptoethanol and stored at ⫺20 °C for at least one night. The lysis of the cells induced by the freezing was completed by sonication. The his-tagged proteins were purified on Ni-NTA resin (Qiagen, Inc.) using standard protocols. The purification of the protein is completed by gel filtration on a Superdex™ 75 column (Amersham Pharmacia Biotech) equilibrated in 50 mM Tris-HCl, pH 8, 200 mM NaCl, 10 mM ␤-mercaptoethanol. The purity of the pooled fractions was checked by SDS-PAGE and the integrity of the final protein samples was confirmed by mass spectrometry. The labelling of the protein with Se-Met was according to standard protocols. Fluorescence Measurements for Binding of Nucleotides and Mant-ATP The nucleotides ATP, ADP, GTP, and N-methylanthranyloyl-ATP (Mant-ATP) were from SIGMA. A SLM Aminco MC200 monochromator was used for the fluorescence measurements. The increase in the extrinsic fluorescence was used to measure the binding of Mant-ATP to YGR205w. Mant-ATP (0 –50 ␮M) was incubated at 20 °C with 3 ␮M YGR205w in 20 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 20 mM ␤-mercaptoethanol. Excitation was performed at 350 nm and emission scanned between 400 and 500 nm with a Cary Eclipse fluoro-spectrophotometer (Varian). The fluorescence of free Mant-ATP (absence of YGR205w) was recorded under the same conditions. Binding of Mant-ATP to YGR205wp was quantified as a difference of fluorescence at 446 nm as a function of protein concentration. In the competition experiments, the protein YGR205wp (3 ␮M) was first incubated with Mant-ATP (2 ␮M). Then, successive aliquots (from 2 to 300 ␮M) of unmodified

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nucleotides ATP, ADP or GTP were added, and the amount of Mant-ATP remaining bound to the protein was monitored by recording residual fluorescence of the Mantgroup. The resulting Lineweaver-Burke plot reveals a competitive inhibition of Mant-ATP binding by ATP and allowed to determine the association constant of ATP. Crystallization and Resolution of the Structure All crystallization trials were performed at 18 °C. Native crystals were grown from a 1/1 mixture of 4 mg/ml protein solution in 25 mM Tris-HCl pH 8 and a reservoir solution of 1.9 M ammonium sulfate, 0.1 M sodium acetate pH 4.5. The crystals belong to the P43212 symmetry space group with cell dimensions a ⫽ b ⫽ 64.25 Å and c ⫽ 140.15 Å. SeMet modified protein crystals were grown from a mixture in a 1/1 ratio of 19mg/ml protein solution in 25 mM Tris-HCl pH 8, 1 mM EDTA and a reservoir solution of 0.2 M ammonium sulfate, 30% PEGMME 2000, 0.1 M sodium acetate pH 4.5. Mono crystals (P43212, a ⫽ b ⫽ 65.26 Å and c ⫽ 140.47 Å) for diffraction measurements were isolated from plate like piles. Both SeMet modified and native proteins crystallized with one molecule per asymmetric unit and the solvent content was 38%. Crystals were prepared for data collection by soaking in crystallization mother liquor containing 20% glycerol. Multiwavelength anomalous diffraction (MAD) data to 2.82 Å resolution were collected from a cryo-cooled crystal (100 K) on the ESRF BM30 beamline. The data were processed using the DENZO and SCALEPACK programs.13 Data sets were processed independently using the SCALA and TRUNCATE programs.14 Merging and scaling of data sets followed the CNS procedure.15 The data collection statistics are summarized in Table 1. The five Se sites were found comparing the predicted and calculated Patterson maps.16 The resulting experimental map was improved by the solvent flipping procedure (implemented in the RESOLVE programme). The initial model was built at a resolution of 2.8 Å using the molecular graphics program TURBO-FRODO (http://afmb.cnrs-mrs. fr/TURBO_FRODO/). Initial refinement was performed on the remote wavelength data set using the maximum likelihood target within the CNS program. At this stage 263 residues out of 290 were build, yielding crystallographic refinement values of Rfree ⫽ 34.61% and R ⫽ 29.78%. The structure was further refined against 20 –2.25 Å resolution data collected from native protein crystals. The final model contains 278 residues and 74 water molecules. Residual density was modelled by 2 sulphate ions (present in the mother liquor) and 3 glycerol molecules (cryo-protectant). Residues 1 to 4 and 117 to 122 were not defined in the 2Fo–Fc electron density map, and are absent from the final model. Coordinates have been deposited in the Protein Data Bank with the accession code 1ODF. RESULTS AND DISCUSSION Overall Structure The structure of YGR205wp was determined by MAD phasing to a resolution of 2.8 Å using Se-Methionine

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I. LI DE LA SIERRA-GALLAY ET AL.

TABLE I. Data-collection and Refinement Statistics Native Wavelength (Å) f⬘/f⬙ space group Unit-cell parameters a, b, c (Å) Resolution (Å) Total number of refl. Total of unique refl. Multiplicity Rmerge1 I/␴(I) Overall completeness (%) Refinement Resolution (Å) Reflections (working/test) Rcryst/Rfree2 Residues Non-hydrogen atoms Water molecules R.m.s. deviation Bonds (Å) Angles (°) Mean B factor (Å2) Protein/solv Ramachandran analysis Most-favored (%) Allowed (%)

0.934

Edge

P43212

0.9796 ⫺10/2.8 P43212

64.9, 64.9, 140.1 30–2.25 94854 14707 6.4 0.055 33.2 98.9

65.3, 65.3, 140.5 99–2.8 183553 7803 11.2 0.062 36.2 98.9

Peak

Remote

0.9794 ⫺7.9/5

0.9719 ⫺4.3/3.8

99–2.8 264666 8000 12.5 0.077 27.5 99.6

99–2.8 184013 7802 11.2 0.059 37.5 98.8

20–2.25 13255/700 0.206/0.26 280 2298 74 0.007 1.195 46.2/42.6 91.8 8.2

Rmerge ⫽ ⌺h⌺i兩Ihi ⫺ 具Ih典兩/⌺h⌺iIhi, were Ihi is the ith observation of the reflection h, while 具Ih典 is the mean intensity of reflection h. 2 Rfactor ⫽ ⌺储Fo兩 ⫺ 兩Fc储/兩Fo兩. Rfree was calculated with a small fraction (4.8%) of randomly selected reflections. 1

Fig. 1. a: Stereo view of the 2Fo–Fc difference electron density map around the region of the P-loop, contoured at 2␴. Clear residual electron density present in front of the P-loop was interpreted as a bound SO4-ion (ball and stick, Sulphur: green, Oxygen: red). b: Ball and stick representation of the SO4-ion bound to the P-loop. c: Ball and stick representation of the SO4-ion bound at the opposite site of the P-loop in the putative phosphate accepting substrate binding groove.

substituted protein crystals. The crystal contains one molecule in the asymmetric unit and according to gel filtration, the protein is a monomer in solution. The structure was refined against 2.25 Å resolution native data. The refined structure yielded a R factor of 20.2% and a Rfree factor of 26%. As illustrated for the region around the P-loop, the electron density was clearly defined for almost all residues (Fig. 1a). All residues are in favorable regions of the Ramachandran plot as defined by PROCHECK.17 All crystallographic refinement data are gathered in Table 1. As illustrated in Figure 2(a), YGR205wp adopts a typical mononucleotide binding fold consisting of a six stranded ␤-sheet (strands order 8-7-6-1-5-2, with strand 8 antiparallel to the five other strands).18 The central sheet is flanked by four alpha-helices (␣1, ␣2, ␣6, and ␣9), two on both sides. Two extra anti parallel strands (␤3 and ␤4) form a small auxiliary sheet that separates from the central ␤–sheet. A long connection between strands 6 and 7, containing two helices (␣7 and ␣8) forms a lid, covering the C-terminal part of the sheet. The P-loop or Walker A motif (G38-X-X-G-X-G-K-S/T) is situated at the connection between strand 1 and helix 2, a characteristic feature of nucleotide binding proteins. The lid and P-loop as well as some of the connections between strands form the walls of a groove that could encompass a putative active site region.

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TABLE II. Structural Closest Neighbours of YGR205wp Protein Panthothenate kinase Phosphoribulokinase 6-phosphofructo-2-kinase Adenylate kinase Thymidylate kinase

PDB code

Z-score

r.m.s. (Å)

Number of compared C␣-positions

Sequence identity (% identity)

1esm 1a7j 1bif 1zin 1g3u

18.1 9.6 9.3 7.9 7.4

2.8 4.1 3.4 3.6 4.1

221 198 160 156 156

15 12 11 14 10

Fig. 3. Alignment of sequences related to YGR205w. Secondary structure elements present in the YGR205wp crystal structure are shown above. Represented sequences are from At: Arabidopsis thaliana (AK258866.1), Te: Trichodesmium erythraeum (gb| ZP00072575.1|), Ns: Nostoc sp (NP486913.1), Sp: Schizosaccharomyces pombe (emb|CAB52731.1), Cc: Caulobacter crescentus (gb|AAK24872.1| ).

Fig. 2. a: Ribbon presentation of the structure of YGR205wp. The central ␤-sheet of the mononucleotide binding fold is shown in yellow, ␣-helices are in blue, the P-loop is in red, the extra ␤-sheet is in green and the lid is in Indian-red. The two bound sulphate ions are shown as ball-and-stick models (yellow for sulphur and red for oxygen). b: Stereo ribbon diagram of the structural superposition of YGR205wp (blue) and pantothenate kinase (red), the structural analogue with the highest Dali Z-score. The sulphate ions belong to the YGR205wp structure and are represented as in part a.

A search for structural homologues with the Dali server,19 as summarized in Table 2 gave good matches with pantothenate kinase (PK),20 phosphoribulokinase (PRK),21 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK),22 adenylate kinase (AK)23 and thymidylate kinase (TK).24 The overall structural superposition of YGR205wp and pantothenate kinase is illustrated in Figure 2(b). Despite a strong structural similarity (Z score 19), the architecture of the active sites of the two proteins is different in the region of the binding site of the phosphate accepting substrate. Yeast has an annotated panto-

thenate kinase (YDR531w), but this ORF has no detectable sequence similarity to YGR205wp. The YDR531w null mutant is non-viable in yeast, suggesting YGR205wp is not functionally compensating. Weaker structural similarity extends over a variety of other enzymes, all of them involved in a phosphate transfer reaction. All these structures have a minimal central five stranded parallel ␤-sheet with identical topology in common. The two most similar structures, pantothenate kinase and phosphoribulokinase have two extra ␤-strands in the central sheet (but YGR205wp has only one) and the auxiliary 2-stranded ␤-sheet extension. Thymidylate kinase, 6-phosphofructo-2kinase and adenylate kinase have a C-terminal helix that occupies the same position as the N-terminal helix of YGR205wp. Apart from the P-loop region and the Walker B motif (I157-L-E-G-W in YGR205wp, Fig. 3), sequence similarity among these proteins is very low. ATP Binding and the Presence of SO4 Ions The presence of the Walker A motif and the structural resemblance of YGR205wp to nucleotide converting en-

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zymes, prompted us to test the binding of nucleotides. We used N-Mant-ATP, a fluorescent ATP analogue, as a reporter group to measure the competitive binding of nucleotides. As can be seen in Figure 4(a), ATP has the highest affinity, binding of ADP is weaker and GTP is not capable of chasing the fluorescent marker. YGR205w binds Mant-ATP in a specific and saturable fashion with a Kd of 9 ␮M (Fig. 4b). This binding was efficiently inhibited in a competitive manner by ATP. Analysis of the displacement curves using an in-house software yielded a Kd of 35 ␮M (⫾ 10 ␮M) for ATP. This value is comparable to what is found for the binding of ATP and ATP analogues to other monunucleotide kinase enzymes.25,26 We conclude YGR205w possesses specificity for the adenine base. Attempts to obtain cocrystals of YGR205wp in the presence of ATP/ADP failed. During refinement of the Se-Met structure (at 2.85 Å resolution, results not shown) , however, clear strong residual electron density became visible for two compounds bound in the putative active-site groove. These residual densities could be modelled by two sulphate ions, present at only 0.2 M in the Se-Met protein crystallization liquor (Fig. 1). Further refinement of these sites was done on the native data at 2.25 Å (crystallized from 1.9 M ammonium sulphate). The two ions occupy niches at the opposite ends of a groove at the C-end of the central ␤–sheet [Fig. 2(a)]. Their interactions with the protein are represented in Figure 1(b).27 The first sulphate ion (SO4-501) is very well defined in the density and has B-factors comparable to those of the surrounding main chain atoms (39 Å2). It is embraced by main chain NHgroups of residues from the P-loop (N-Gly41, N-Gly43, N-Lys44 and N-Ser45) and forms an additional H-bond with the O␥ from Ser45 and a salt bridge with Lys44. The second sulphate ion (SO4-502) is bound at the opposite side of the groove, at 10 Å from the first sulphate ion, and it has higher B factors (60 Å2). It forms a salt bridge with Arg96 (projecting from the irregular connection between ␤2 and ␤3) and Lys133 (projecting from the auxiliary ␤–sheet) and a hydrogen bond with the hydroxyl of Tyr74 (situated at the exit of ␤2). The position of these sulphate ions in a groove formed by the P-loop, the lid and the strand connections, outline very likely a substrate binding site in YGR205wp. The roof of this groove is determined by the aliphatic part of Gln40, and a hydrophobic patch containing Tyr248 and Phe244. The floor of the groove is hydrophobic (Trp161) and the rim opposite to the P-loop is decorated by charged side chains (Lys44, Asp71, Lys33, Arg66). The SO4-501 ion in YGR205wp superposes with the ␤-phosphate position of bound ATP-compounds in the active sites of PK, PFK and AK, and also with a SO4 ion bound near the P-loop in the TK crystal structure.24 The interactions with the P-loop of SO4-1 in YGR205wp and of the nucleotide phosphate groups in the PK and PFK complexes are very similar.20,21 The superposition of the YGR205wp structure with its structural neighbors provides us insights about the possible binding modes of ATP and phosphate acceptor substrates. We compared the putative active site of YGR205wp with those of pantothenate kinase, phosphoribulokinase,

Fig. 4. a: Displacement titration experiment of Mant-ATP bound to YGR205w with unmodified nucleotides. Upon excitation at 356 nm, Mant-ATP binding to YGR205w was monitored by the increase in the fluorescence intensity of the Mant-group at 440 nm. Unmodified nucleotides used as competitors were ATP (circles), ADP (triangles) or GTP (squares). b: The binding of Mant-ATP to YGR205w was measured by fluorescence titration as described in Materials and Methods. The increase of extrinsic fluorescence of Mant-ATP in presence of the protein was plotted versus Mant-ATP concentration. Kd value was calculated using a hyperbolic regression analysis program (MDFIT).

6-phosphofructo2-kinase, adenylate and thymidylate kinases. The active sites of all these enzymes have a common architecture: the P-loop region that forms the nucleotide binding site, a flexible lid region and a pocket providing the binding site for the phosphate accepting substrate. In the structure of YGR205wp, the lid contains helices ␣7, ␣8 and ␣9 and the pocket is formed by ␣3, ␣6 and the loops that connect these helices to the core region. The nucleotide (phosphate donor) binding site is similar, but the relative position of the lid and the second (phosphate acceptor) substrate binding sites are very different in all the enzymes. The variable nature of the second site reflects the very different phosphate-accepting substrate specificities of these enzymes. In order to get insight into the binding mode of ATP to YGR205wp we superposed its structure with that of a complex between PK and AMPPNP. A local structural alignment between YGR205wp and PK focussed on the


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Fig. 5. The YGR255w and the pantothenate kinase Ca traces are in green and brown respectively. The YGR255w and pantothenate kinase P-loops are in yellow and orange, respectively. The YGR255w residues are represented as sticks and labelled in bold, those of pantothenate kinase are depicted in ball and sticks and labelled in italics. The Mg2⫹ ion bound the AMPPNP is in cyan.

P-loop region is represented in Figure 5. AMPPNP in PK is bound in a groove contained between the P-loop, the loop preceding helix ␣1, and the connecting loop between strands ␤9 and ␤10. The adenine base and ribose ring make few direct specific interactions. The base ring is sandwiched between the side chains of Asn43 and His307. The groove in YGR205wp that corresponds to the ATP binding site in PK, AK, or TK is much more accessible to solvent. There are two main reasons for this: (1) the lid region and loops that interact with nucleotide adopt a more open conformation in YGR205wp and (2) the Nterminus that also contributes to the adenine binding site in PK is truncated in YGR205wp. The conformation of the P-loop in the YGR205wp structure is well adapted to engage in interactions with the phosphate groups of ATP as seen in other mononucleotide binding proteins, but the remainder of the nucleotide will probably be stabilized only after conformational changes in the region of the adenine base. In structures of complexes of PK and PFK with ATP analogues, the nucleotide is providing two oxygens (one on the ␤ and one on the ␥ phosphate groups) to an essential Mg ion that is also bound to the protein. The coordination is completed by a OH group from threonine/serine (Thr52 in PFK, Ser102 in PK) and a carboxylate from Glu/Asp (Asp128 in PFK and Glu199 in PK). YGR205wp has the same residues at these positions (Ser45, Glu159) and should therefore be able to bind an ATP-Mg complex.

From modelling studies it was proposed that Asp127 in PK could be involved in nucleophilic activation of the substrate hydroxyl group.20 Site-directed mutagenesis of an equivalent aspartate in PRK has demonstrated its importance in catalysis for that enzyme.28 An aspartate side chain (Asp71) is found in an equivalent position in our YGR205wp crystal structure (Fig. 5). It was also proposed that Arg243 in PK is ideally positioned to stabilize the negative charge that develops in the trigonal bipiramidal transition state of the ␥-phosphate group. Site-directed mutagenesis studies of the equivalent arginine in PRK resulted in a reduction of enzymatic activity.29 YGR205wp has an arginine in a similar position (Arg221) that is part of the region that shows high flexibility and that zooms in onto ATP in the various structures. This arginine is totally conserved in all the related sequences (Fig. 3). All these facts indicate that YGR205wp could be a small metabolite kinase. Nucleoside monophosphate kinases can adopt an open structure in the absence of substrate, a partially closed intermediate with a single substrate bound and a fully closed form in the presence of both substrates.26 The size, nature and orientation of the binding pocket of the phosphate acceptor substrate is very different among the various enzymes, making a precise structural comparison difficult. This is due to (1) the very different configurations of the loops lining this pocket and to (2) the flexibility of the lid and phosphate acceptor substrate binding region, exemplified for instance in the various adenylate kinase struc-

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tures.30 In the structure of YGR205wp we observed that a sulphate binds in the acceptor pocket, interacting mainly with charged/polar side chains. As can be seen in Figures 1(b) and 3, all of the residues interacting with this sulphate ion (Tyr74, Gly96, Lys97, and Lys133) are strictly conserved in the close sequence relatives of YGR205wp, suggesting the ion may occupy the position of a phosphate ion on the substrate. The crystal structure of PRK has a disordered P-loop but shows a sulphate ion that is bound in a closely related position to SO4-502 in YGR205wp. This site was proposed to be the ribulose-5-phosphate binding site in PRK.21 Another enzyme, TK, has TMP bound in this pocket and the phosphate is in a similar position as the sulphate group in YGR205wp. Rough modelling of potential acceptors in the YGR205wp pocket, shows that its volume is suited for binding a molecule of the size of phosphoribulose, but not considerably bigger. Superposition with TK and PK for instance clearly indicates that this pocket in YGR205wp is not shaped for accepting substrates of the size of thymidine or pantothenate (not shown). This assumes that the volume of the pocket in YGR205wp does not change significantly upon ligand binding. At this stage however it is not possible to make further projections on the nature of the phosphate acceptor and hence on the biochemical function of YGR205wp. CONCLUSION The structure of YGR205wp presents a typical mononucleotide-binding fold and shows the strongest structural resemblance to the pantothenate kinase of E. coli. The crystal structure of YGR205wp has two sulphate ions bound in the potential active site groove. The first one corresponds to the phosphate position of a P-loop bound nucleotide in other enzymes of this type, indicating the YGR205wp may indeed be a small metabolite kinase. Comparison with other structurally similar kinases shows us that YGR205wp possesses all the functional groups for catalyzing a transfer of a phosphate group to a yet unknown acceptor. The second sulphate binds at the opposite site of the binding groove. This sulphate suggests that a phosphate group may be present on the phosphate acceptor substrate. The described YGR205wp crystal structure probably represents an open, inactive conformation of the protein. This study shows both the strength and limitations of structural genomics for the discovery of protein function. The structure gives strong hints for molecular function but further biochemical experiments are needed to test and to confirm these hypotheses. ACKNOWLEDGMENTS This work is supported by grants from the Ministe`re de la Recherche et de la Technologie (Programme Geńopoles) and Association pour la Recherche contre le Cancer (to M Graille). The authors acknowledge staff from the ESRF beamlines for help with data collection. REFERENCES 1. Mewes HW, Albermann K, Bahr M, Frishman D, Gleissner A, Hani J, Heumann K, Kleine K, Maierl A, Oliver SG, Pfeiffer F, Zollner A. Overview of the yeast genome. Nature 1997;387(6632

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