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In order to extend the structural coverage of eukaryotic members in the Protein Family database (PFAM),1 we selected 400 open reading frames (ORF's) from the Mouse genome from available cDNA libraries. One of these, the mouse gene GI: 18204011 belongs to the structurally uncharacterized subfamily KOG11962 of zinc-containing dehydrogenases (PF00107),3 which has over 2000 homologues in all kingdoms of life. Thus, the 18204011 gene of mouse encodes a putative NADPH-dependent oxidoreductase, with a molecular weight of 37,921 Da (residues 1–351) and a calculated isoelectric point of 5.4. Here, we report the crystal structure of this putative oxidoreductase determined using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4

The crystal structure of 18204011 [Fig. 1(A)] was determined to 2.10 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one protein monomer (residues 1–253, 266–351) and 153 water molecules. No electron density was observed for residues 254–265. The Matthews' coefficient (Vm)5 for 18204011 is 2.60 Å3/Da and the estimated solvent content is 51.8%. The Ramachandran plot, produced by Procheck 3.4,6 shows that 93.7% of the residues are in the most favored regions and 6.3% are in additional allowed regions.

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Figure 1. Crystal structure of 18204011. A: Stereo ribbon diagram of mouse 18204011 color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization and location of the putative active site (arrow). Helices H1–H17, and β-strands (β1–β15) as well as β-sheets A, B, C and beginning (C253) and end (P266) of the disordered loop are indicated. B: Diagram showing the secondary structure elements in 18204011 superimposed on its primary sequence. The strands in each β-sheet are indicated by a red A, B, and C. β-hairpins are depicted as red loops. Disordered regions are depicted by a dashed line with the corresponding sequence in brackets. β-bulges are marked by β; γ-turns are marked by γ.

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Table I. Summary of Crystal Parameters, Data Collection, and Refinement Statistics for 18204011 (PDB: 1vj1)
  • a

    Highest resolution shell.

  • b

    Rsym = Σ|Ii − Ii|/Σ|Ii| where Ii is the scaled intensity of the ith measurement, and Ii is the mean intensity for that reflection.

  • c

    Rcryst = Σ| |Fobs| − |Fcalc| |/Σ|Fobs| where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.

  • d

    Rfree = as for Rcryst, but for 5.0% of the total reflections chosen at random and omitted from refinement.

  • e

    ESU = Estimated overall coordinate error.14, 18

Space groupP212121
Unit cell parametersa = 42.39 Å, b = 91.81 Å, c = 100.57 Å, α = β = γ = 90°
Data collectionλ1MADSeλ2MADSeλ3MADSe
Wavelength (Å)0.97950.95670.9793
Resolution range (Å)50.28–2.10100–50.3550.39–2.60
Number of observations135,15799,70590,496
Number of reflections22,43315,7.1012,402
Completeness (%)95.3 (74.9)a98.5 (99.7)a98.3 (99.6)a
Mean I/σ(I)10.9 (1.7)a11.5 (2.8)a9.8 (1.9)a
Rsym on Ib0.069 (0.431)a0.074 (0.351)a0.087 (0.495)a
Sigma cutoff0.00.00.0
Highest resolution shell (Å)2.15–2.102.46–2.402.67–2.60
Model and refinement statistics   
Resolution range (Å)50.28–2.10Data set used in refinementλ1MADSe
Number of reflections (total)22,433Cutoff criteria|F| > 0
Number of reflections (test)1123Rcrystc0.180
Completeness (% total)95.2Rfreed0.216
Stereochemical parameters   
Restraints (RMS observed)   
Bond length0.016 Å  
Bond angle1.47°  
Average isotropic B-value19.6 Å2  
ESU based on R valuee0.20 Å  
Protein residues/atoms341/2,567  
Solvent molecules153  

The 18204011 monomer contains 15 β-strands (β1–β15), eight α-helices (H6, H7–H11, H13, H14), and nine 310-helices (H1–H5, H7′, H12, H13′, H14′) [Fig. 1(A, B)]. The total β-strand, α-helical, and 310-helical content is 29.0%, 22.6%, and 7.9%, respectively. The 18204011 structure is a member of the ζ-crystallin subfamily within the medium-chain dehydrogenase/reductase (MDR) superfamily.3 It comprises two distinct domains [Fig. 1(A, B)]: the catalytic domain (residues 1–132; 311–351) and the nucleotide-binding domain (residues 133–310). The catalytic domain contains nine β-strands arranged in two β-sheets (A and B) and six helices (H1–H5; H14). β-sheet A is antiparallel and composed of strands β1 and β2. β-sheet B is a highly twisted, seven-stranded (β3–β7, β14, β15) and forms a partly open, β-barrel-like structure with 67′41235 topology. Strands β3– β7 and β15 are antiparallel, whereas strand β14 is parallel to β15. Helices H5 and H13 to H14 form linker regions between the two domains.

The nucleotide-binding domain folds into a three-layer αβα structure with a classical Rossman-fold. The domain comprises six β-strands (β8– β13) and eight helices (H6–H13), arranged as a six-stranded parallel β-sheet C, with 321456 topology, flanked by six α-helices. The domain contains an unusual nucleotide binding motive 162-GXXGXXG-168, with the GXGXXG being a much more common fingerprint of nucleotide binding. The present structure does not contain a bound nucleotide, which is most likely the reason for the observed disorder in one of the nucleotide-recognition loops (residues 254–265). In contrast to its annotation as a zinc-containing dehydrogenase, the 18204011 structure does not contain any zinc-binding residues that could form a metal center in its putative active site. The crystallographic packing in the 18204011 structure suggests that a monomer is the biologically-relevant form.

A structural similarity search, performed with the coordinates of 18204011 using the DALI server,7 indicates structural similarity to a zinc-independent quinone oxidoreductase from E. coli (PDB: 1qor),8 with an RMSD of 3.4 Å over 326 aligned residues with 18% sequence identity [Fig. 2(A)]. Another structural homologue is enoyl thioester reductase (Etr1p) from Candida tropicalis (PDB: 1guf),9 where the RMSD is 3.4 Å over 305 aligned residues with 19% sequence identity.

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Figure 2. A: Ribbon diagram of a superposition of 18204011 (mouse) and quinone oxidoreductase from E. coli (PDB: 1qor) grey. The structures were superimposed on their nucleotide-binding domains. The NADPH molecule bound to quinone oxidoreductase is shown in cpk mode. B: Close up view of the active site. The NADPH molecule and the sulfate bound to the active site of quinone oxidoreductase are shown in ball and stick. The active site tyrosine (Y52) as observed in quinone oxidoreductase from E. coli and its potential counterpart (Y64) in 18204011 (the Y64 side-chain has been modeled here due to disorder in the crystal structure) are shown in ball and stick.

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A detailed structural comparison with quinone oxidoreductase, which also does not contain zinc-coordinating residues, shows that the large cleft between the two domains in 18204011 could easily accommodate an NADPH molecule [Fig. 2(A, B)]. The relative disposition of the catalytic domains in the two structures shows that NADPH binding in quinone oxidoreductase is accompanied by a conformational change in the hinge region which moves the two domains closer to each other. This rigid body movement is observed in comparisons of the structures of the apo and NADPH-bound forms of quinone oxidoreductase.8 18204011 also contains positively-charged Lys192, as well as Tyr208, for the interaction with the phosphate group of NADPH. 18204011 also contains Gly 188, which is characteristic for proteins, exhibiting NADP rather than NAD specificity. The disordered loop between residues Cys253 and Pro266 comprises a conserved GxxS motif, which stabilizes both the adenine and nicotinamide moieties of the cofactor in the NADPH-bound form of quinone oxidoreductase. The analogy to quinone oxidoreductase indicates that 18204011 also binds NADPH and that its active site is also located in the groove next to helices H2, H5, and H6, which are adjacent to the nicotinate moiety of the bound NADPH molecule. The hydroxyl group of a catalytic tyrosine (or serine) acts as an electrophilic catalyst in the enzymatic reaction of quinone oxidoreductase and Etr1p.8, 9 18204011 contains Tyr64 [Fig. 2(B)], a potential counterpart for the catalytic tyrosine residue in quinone oxidoreductase (Tyr52) and enoyl thioester reductase (Tyr79). The Tyr64 side-chain is disordered in the 18204011 structure, which is most likely due to lack of any bound NADPH. Tyr64 appears to be conserved in the closest sequence homologues of 18204011 across all kingdoms of life, which suggests a redox mechanism related to that of quinone oxidoreductase.8 However, the reaction catalyzed by 18204011, awaits further biochemical studies and identification of its substrate and products.

According to the Fold and Function Assignment System (FFAS),10 the subfamily including 18204011 has about one hundred homologous sequences in eukaryotic proteomes. Models for 18204011 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=18204011.

The 18204011 structure reported here represents a putative NADPH-dependent oxidoreductase from mouse, whose structure has been determined by X-ray crystallography using the MAD method. The information reported here, in combination with further biochemical and biophysical studies will yield valuable insights into the functional determinants of this protein in mammals.

Materials and Methods.

  1. Top of page
  2. Materials and Methods.
  3. Acknowledgements
  4. REFERENCES

Protein production and crystallization:

A putative NADPH-dependent oxidoreductase from Mus musculus (GI: 18204011, IMAGE: 5068419, Swissprot: Q8VDQ1) was amplified by PCR from a clone obtained from the IMAGE consortium using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH4, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/ml. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer (50 mM Tris, pH 7.9, 50 mM NaCl, 10 mM imidazole, 0.25 mM Tris[2-carboxyethyl]phosphine hydrochloride [TCEP]), and cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-resin (Amersham Biosciences) pre-equilibrated with Lysis Buffer. The nickel-resin was washed with Wash Buffer [50 mM potassium phosphate, pH 7.8, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP], and the protein eluted with Elution Buffer [20 mM Tris, pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer A [20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) pre-equilibrated with Buffer A. The protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer A. Appropriate fractions were further purified using a Superdex 200 column (Amersham Biosciences) with isocratic in crystallization buffer (20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP). The protein was concentrated for crystallization assays to 18 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method11 with standard Joint Center for Structural Genomics crystallization protocols.4 The crystallization solution contained 15% polyethylene glycol (PEG) 4000, 0.2 M NH4-acetate, and 0.1 M Na-citrate (pH 5.1). The cryo-solution contained 15% ethylene glycol in addition. The crystals were indexed in the orthorhomic space group P212121 (Table I).

Data collection.

Anomalous diffraction data were collected at the Advanced Light Source (ALS, Berkeley, USA) on beamline 8.2.2 at wavelengths corresponding to inflection point (λ1), high energy remote (λ2), and peak (λ3) of a selenium MAD experiment, using the BLU-ICE12 data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 CCD detector. Data were integrated and reduced using MOSFLM13 and then scaled with the program SCALA from the CCP4 suite.14 Data statistics are summarized in Table I.

Structure solution and refinement.

The structure was determined by using the CCP4 suite14 and SOLVE/RESOLVE.15 Structure refinement was performed using REFMAC5,14 O,16 and Xfit.17 Refinement statistics are summarized in Table I. The final model includes one protein monomer (residues 1–253, 266–351), two histidine residues from the purification tag, and 153 water molecules in the asymmetric unit. No electron density was observed for residues 254–265, the side-chain atoms of D32, Y64, E115, E191, N212, R286, E297, K320, E347, or the rest of the expression and purification tag.

Validation and deposition.

Analysis of the stereochemical quality of the models was accomplished using PROCHECK 3.4 and SFcheck 4.0.7, 14 Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/) and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of 18204011 have been deposited within the PDB and are accessible under the code 1vj1.

Acknowledgements

  1. Top of page
  2. Materials and Methods.
  3. Acknowledgements
  4. REFERENCES

This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a National user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).

REFERENCES

  1. Top of page
  2. Materials and Methods.
  3. Acknowledgements
  4. REFERENCES