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1 Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-Unité Mixte de Recherche (CNRS-UMR 8619) and
2 Institut de Génétique et Microbiologie (CNRS-UMR 8621), Université Paris-Sud, 91405 Orsay, France
3 Laboratoire d’Enzymologie et Biochimie Structurale (CNRS-UPR [Unité Propre de Recherche] 9063), 91198 Gif sur Yvette, France
4 Centre de Bioingénierie Gilbert Durand, UMR-CNRS 5504, UR-INRA 792, F-31077, Toulouse, France
Reprint requests to: Herman van Tilbeurgh, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire (CNRS-UMR 8619), Université Paris-Sud, Bât. 430, 91405 Orsay, France; e-mail: Herman.Van-Tilbeurgh{at}ibbmc.u-psud.fr; fax: +00-331-69-85-37-15.
(RECEIVED September 14, 2004; FINAL REVISION September 16, 2004; ACCEPTED September 16, 2004)
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Abstract |
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 TOP Abstract IntroductionResults and DiscussionMaterials and methodsReferences |
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-strands organized in two parallel packed -sheets. The protein has strong structural resemblance to the plant storage and ligand binding proteins (canavalin, glycinin, auxin binding protein) but also to some plant and bacterial enzymes (epimerase, germin). The protein forms homodimers in the crystal, confirming measurements of its molecular mass in solution. Two monomers have their -sheet packed together to form the dimer. The presence of a hydrophobic ligand in a well conserved pocket inside the barrel and local sequence similarity with bacterial epimerases may suggest a biochemical function for this protein. Keywords: jelly-roll motif; cupin superfamily; structural genomics; YML079wp; S. cerevisiae
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041121305.
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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Although we prioritized our targets for the discovery of new folds, the protein structures solved in our project inevitably brought us to investigate about their biochemical function. Structure might indeed reveal features undetectable by sequence analysis: (1) analogies with proteins of known function and (2) the presence of functional sites (Stark and Russell 2003). In these favorable cases structure provides a hypothesis for biochemical function, testable through site-directed mutagenesis and cocrystallization. Sometimes the crystal structure reveals the presence of a fortuitous ligand in an unexpected binding pocket providing some hints on the binding specificity of the protein (Zarembinski et al. 1998; Liger et al. 2004). The precise biochemical and cellular functions of the protein remain, however, frequently unknown and firm confirmation necessitates further experimental investigation (Graille et al. 2004).
The yeast YML079w gene codes for a hypothetical protein of 201 amino acids (molecular weight 22,460 Da and a calculated isoelectric point of 5.2) of unknown function. The sequence has similarity with a number of bacterial open reading frames (sequence identity around 30%). Homologous genes are also present in the eukaryotic organisms Neurospora crassa and Arabidopsis thaliana (35% sequence identity). None of these sequences is related to a protein of known function. Threading protocols do not propose a reasonable structure prediction for this ORF. Since the structure and function of this protein are unknown, it provided an excellent target in our structural genomics project. We determined the crystal structure of this protein by single wavelength anomalous scattering measurements on Semethionine substituted crystals and refined it to 1.75 Å resolution. The structure of YML079wp defines a new sequence family of the well-known vivilin/germin jelly-roll fold.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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). However, they suffered from severe mosaic and anisotropic diffraction and hence could not be exploited for the resolution of the structure. Crystals with improved mosaicity were obtained in the presence of ATP (a ligand in our optimization screens) in a new crystal form (I432). Although diffraction of these crystals is weak and resolution reaches only to 2.8 Å, the high symmetry space group allowed the collection of very redundant anomalous single wavelength data on crystals grown from SeMet substituted protein. The excellent quality of the single wavelength anomalous diffraction (SAD) maps allowed easy construction of the model. Most of the protein structure was well defined, but no electron density was observed for the five residues at the N terminus and a short connection between two -strands (residues 65–69). Electron density is missing for these regions in the three independent copies of the molecule in the crystal, suggesting this is due to inherent mobility. The three molecules in the asymmetric unit have identical structures (RMSD between the molecules: 0.37 Å). As measured by analytic gel filtration, YML079wp forms a dimer in solution (results not shown). Analysis of packing in the crystals shows that two subunits form a tight homodimer related by a local twofold axis and the third protomer is part of second dimer that is generated by a regular crystal symmetry axis.
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, the structure of the protein consists of 12 -strands and three 
-helices. Ten -strands are part of two five-stranded anti-parallel -sheets (S1, strands 141169 and S2, strands 1251078), packed parallel against each other and forming a -barrel known as jelly-roll motif. Two extra strands, 2 and 3 are connected by the mobile region and form a handle-like extension from the roll. The N-terminal part of the molecule starts with an extended conformation that covers the back of -sheet S2, followed by helix 1. At the C-terminal part, a small two-helical anti-parallel bundle lies against the back of sheet S2.
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. All these proteins share a very similar jelly-roll core. None of the structural homologs have the -sheet handle extension as observed in YML079wp. The N-and C-terminal regions that are not part of the jelly-roll motif are also very different between these proteins and YML079wp.
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). Dimer formation buries 2680 Å2 (i.e., 13.5% of the accessible surface area), a value very similar to what was observed for other dimers of this structural family. The dimer involves 26 residues from each molecule and is stabilized by 13 hydrogen bonds, 2 salt bridges, and packing of hydrophobic residues (Fig. 1B). In YML079wp structural homologs such as dTDP-4-dehydrorhamnose 3,5-epimerase (hereafter named RmlC), canavalin, and auxin-binding protein, homodimerization occurs through the same region and the resulting homodimers can be superimposed onto YML079wp with RMSD around 3 Å (Table 2). The YML079wp residues responsible for homodimer formation are not conserved within RmlC and auxin binding protein and even poorly conserved among the closest YML079wp sequence homologs (Fig. 2). Hence, although members of the cupin superfamily form similar quaternary structures, the residues involved in these are not well conserved.
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) shows a number of strictly conserved residues. Six of these (Gly45, Gly46, Ile82, Tyr83, Leu85, and His96) are situated in a pocket lying between the two sheets of the central barrel (strands 1, 4 and 5; Fig. 3A). The entrance of the YML079wp pocket forms a hydrophobic seal, while the bottom contains in majority polar/charged residues. Global superposition of YML079wp onto auxin-binding protein, epimerases (RmlC and EVAD), phosphogluco mutase and germin, shows that this pocket coincides with the substrate/ligand binding sites of all these proteins.
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). Clear density for guanine was observed in the aforementioned pocket and the base occupies the same site as for the ATP complex (Fig. 3B). We could only model the guanine base of GTP; no density was present for sugar nor phosphate groups. Five residues from YML079wp are in close contact with the ligand. The guanine ring is stacked between Phe48 and Ile82 on one side and Phe171 on the other and forms two hydrogen bonds with side chains from His42 and Glu50. Four of these residues are strictly conserved in YML079wp homologs as well as the aromatic ring of the fifth (Phe48 replaced by Tyr in some homologs; Fig. 2). Residual electron in close proximity to this guanine has been modeled by an acetate molecule.
The best structural match of this YML079wp base binding pocket is found with the nucleotide binding pocket of RmlC. However in YML079wp, all the -strands contributing to the pocket are from the same monomer, while in the available RmlC structures, these strands come from different monomers (Christendat et al. 2000; Giraud et al. 2000). The amino acid stretch at the active site entrance shows some degree of conservation. The RmlC family is characterized by the presence of a 24RG
FE29 signature (where is a hydrophobic residue, numbering according to Salmonella enterica RmlC). In RmlC structures, this peptide is localized in strand 3, which through its interaction with a strand from the other monomer, extends the RmlC -sheet responsible for homodimer formation. This RmlC strand coincides with YML079wp strand 1 that bears the conserved sequence 45GG(R/K)E50 (Fig. 2). Two residues from this peptide seem to play a critical role in ligand binding. First, the aromatic side chain of the RmlC conserved Phe27 and of the YML079wp Phe48 (according to YML079wp numbering) is involved in stacking with the base moiety of the respective ligands. Second, the glutamate at positions 29 in RmlC and 50 in YML079wp is hydrogen-bonded to a nitrogen atom from the thymidine or the guanine bases, respectively.
Further analysis of the superposition between the YML079wp and the S. suis RmlC complexed to a substrate analog shows that the RmlC sugar moiety binding cavity (Dong et al. 2003), is also present in YML079wp (Fig. 3C). In RmlC, this cavity harbors the residues critical for enzymatic activity and/or substrate binding: His63, Lys73, Tyr133, and Asp170 (Salmonella enterica numbering). RmlC His63 matches with YML079wp His96, which is totally conserved in all the structural homologs, even those with no documented catalytic activity such as auxin-binding protein, germin, or canavalin. This YML079wp cavity is made by well-conserved residues (His42, His96, Tyr84) and also coincides with the auxin-binding pocket. A carboxylate group (Glu44), which is almost totally conserved in the related sequence family is situated close to His96 (Fig. 2). No strictly conserved residues corresponding to RmlC Lys73 and Tyr 133 are present. We were not able to demonstrate epimerase activity on a set test UDP-substrates. In conclusion, the discussed pocket in YML079wp is very likely involved in binding of a ligand that remains to be identified, but that probably contains an aromatic group. The pocket also has some but not all characteristics of active site pockets of a few enzymes within the cupin superfamily.
Conclusion
The structure of YML079wp shows that it is a new member of the jelly-roll fold superfamily and hence we propose to annotate the gene as CFF1 for Cupin Fold Family 1. This fold is already observed in a number of plant storage proteins and enzymes from plant and bacterial origin. All these proteins share a conserved surface pocket that in many cases has been identified as substrate or ligand binding sites. Sequence analogies with RmlC epimerase in the base binding pocket and conservation of a catalytic base (His96) suggest that YML079wp could be an enzyme involved in the modification of nucleotide sugars. This hypothesis is tempered, however, by the fact that some conserved residues are also present in storage proteins that have no documented ligand binding and enzymatic capacities. A number of epimerase reactions on nucleotide sugars are known in yeast for which no enzyme was associated yet. This hypothesis is under investigation for YML079wp.
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Materials and methods |
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Crystallization and resolution of the structure
Se-Met substituted protein crystals were grown from a mixture of equal volumes of 9 mg/ml protein solution in 20 mM Tris-HCl (pH 8), 10 mM –mercaptoethanol, 100 mM sodium chloride and a reservoir solution of 20%–24% PEG4000, 0.1 M Na-citrate (pH 5.6) and 0.2 M ammonium acetate. For data collection, crystals were flash-frozen after transfer into the crystallization solution containing 30% glycerol. The protein crystallized in the cubic space group I432 (a = b = c = 208 Å) with three subunits per asymmetric unit and the solvent content was 55%. Single wavelength anomalous diffraction (SAD) data to 2.8 Å resolution were collected from a crystal flash cooled at 100 K on the ESRF BM30A beam line in Grenoble. The data were processed using the MOSFLM and SCALA programs from the CCP4 package. The nine Selenium sites were found by Patterson methods with the program SOLVE (Terwilliger and Berendzen 1999). The experimental map was improved by solvent modification within the program RESOLVE (Terwilliger and Berendzen 1999). The resulting map was of very good quality and most of the protein model (181 out of 201 residues) was built using the molecular graphics program TURBO-FRODO (http://afmb.cnrs-mrs.fr/TURBO_FRODO/). Refinement was performed with the maximum likelihood target using amplitudes and experimental phases within the CNS program (Brunger et al. 1998) followed by manual checking and model rebuilding using TURBO-FRODO. The final model yielding crystallographic refinement values of R = 22.3% and Rfree = 28.4% contains 570 residues provided by three protein chains and 180 water molecules. After refinement, strong residual density remained in a pocket on the surface between two aromatic residues. The flatly shaped electron density was modeled as an adenine base because ATP was present in the crystallization solution. Residues 65 to 68 and five amino-terminal residues were not defined in the 2Fo-Fc electron density map, and are absent from the final model. The statistics on data collection, phasing and refinement are summarized in Table 1.
In order to establish any nucleotide preference, we cocrystallized the native protein in presence of many different nucleotides. Good diffracting crystals were obtained with 5 mM GTP in the reaction mixture. They diffracted to 1.7 Å resolution on the ESRF BM30A beam line. The data were processed using the XDS program (Kabsch 1993). As observed for the crystals grown in the presence of ATP, the space group is I432 with three molecules in the asymmetric unit. The structure was refined with the CNS program to 1.75 Å resolution, followed by manual checking and model rebuilding using TURBO-FRODO. Clear residual electron density was modeled as the guanine moiety of the GTP introduced in the crystallization assays. Although resolution was higher, the residues which were not present in the ATP-bound form could not have been modeled into the electron density maps. The atomic coordinates and structure factors for the YML079wp GTP and ATP-bound structures have been deposited into the Brookhaven Protein Data Bank under the accession numbers 1XE7 and 1XE8, respectively.
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Footnotes |
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6 These authors contributed equally to this work.
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References |
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