The R-free was calculated using a subset of 5% of randomly selected reflections.
Structural genomics of Caenorhabditis elegans: Structure of dihydropteridine reductase
Article first published online: 22 OCT 2003
Copyright © 2003 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 53, Issue 4, pages 944–946, 1 December 2003
How to Cite
Symersky, J., Li, S., Carson, M., Luo, D., Luan, C.-H. and Luo, M. (2003), Structural genomics of Caenorhabditis elegans: Structure of dihydropteridine reductase. Proteins, 53: 944–946. doi: 10.1002/prot.10482
- Issue published online: 17 NOV 2003
- Article first published online: 22 OCT 2003
- Manuscript Accepted: 10 APR 2003
- Manuscript Received: 7 APR 2003
The biosynthesis of catecholamines1–3 includes hydroxylation of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Corresponding hydroxylases use tetrahydrobiopterin (BH4), which is oxidized to a quinonoid form of dihydrobiopterin (qBH2). Dihydropteridine reductase (DHPR) then uses NADH as a cofactor to reduce qBH2 back to BH4. The reaction consists of a protonation of the qBH2 substrate and a hydride transfer from NADH.
DHPR from various mammalian sources are very similar and have been isolated as dimers with molecular weight of 25 kDa per subunit. There are only 10 conservative amino acid differences between the rat and human DHPR. The crystal structures of rat DHPR4, 5 and human DHPR6 have been determined as binary complexes with NADH. DHPR is an α/β protein with a central β-sheet surrounded by a layer of α-helices. The first six strands of the β-sheet, together with connecting α-helical segments, have the topology of the dinucleotide-binding fold.7 Dimerization of DHPR is mediated by a four-helix bundle with an unusual right-handed twist.
Here we report the high-resolution structure of an apo form of dihydropteridine reductase from Caenorhabditis elegans (ceDHPR) as a part of the Structural Genomics of C. elegans project.8 The sequence of ceDHPR has 43% identity with human DHPR and 49% identity with rat DHPR. Although crystallized as an apo enzyme, ceDHPR has a remarkable structural homology with mammalian DHPR/NADH complexes. Binding of NADH apparently does not elicit any major conformational changes in the molecule of DHPR. Binding mode of the substrate qBH2 remains hypothetical, assuming the same conformation of the active site to allow protonation of the substrate from the bulk solvent region.
The protein expression and purification are reported in detail at the web site http://sgce.cbse.uab. edu. An intact ceDHPR protein without the histidine tag was released by enzymatic digestion with thrombin. The protein stock solution was concentrated to 17.3 mg/mL in 10 mM HEPES, pH 7. Screening for crystallization conditions was performed by using the commercial kits Wizard I (Emerald Biostructures) and Natrix (Hampton Research). Crystals were grown at 295 K by vapor diffusion in hanging drops. The reservoir solution consisted of 20% (w/v) PEG8000, 10 mM MgCl2, 0.1 M ammonium sulfate, 50 mM MES, pH 5.6. Drops were made of 2 μL of protein stock solution and 4 μL of the reservoir solution. Crystals were diffracted on a home source to 1.8 Å and were characterized as triclinic, space group P1, a = 41.926 Å, b = 50.892 Å, c = 58.77 Å, α = 89.98°, β = 71.98°, γ = 82.05° and with two crystallographically independent molecules.
The crystals were flash-frozen in liquid nitrogen, and native diffraction data to 1.65 Å resolution were collected at 100 K from one crystal at the wavelength 1.07175 Å (Table I). Diffraction images were processed in HKL2000,9 and the initial phases were obtained by molecular replacement in Amore10 using one molecule of the rat DHPR as a search model [Protein Data Bank (PDB) code 1DHR]. Further model building was performed in O,11 and the structure was refined in the Crystallography and NMR system (CNS).12 A number of alternate conformations of side-chains were found in both subunits and refined also in CNS. Table I shows the refinement statistics.
|Resolution (last shell)[Å]||32.28–1.65 (1.71–1.65)|
|Rsym (last shell) [%]||4.1 (28.1)|
|Completeness (last shell) [%]||95.3 (93.8)|
|No. of observations||102854|
|R-factor (R-free)* [%]||19.4 (22.7)|
|No. of protein atoms||3488|
|No. of water sites||601|
|No. of MES atoms||24|
|Average B-factor [Å2]||16.1|
|RMSD bond lengths [Å]||0.004|
|RMSD bond angles [°]||1.2|
|most favorable [%]||92.8|
Except for N-terminal methionines, the final model has two complete ceDHPR chains and includes 470 amino acid residues, 601 water sites, and two molecules of MES. The conventional R-factor is 19.4% and R-free is 22.7% using no cutoffs. The structure has been validated in Whatcheck13 and deposited in the Protein Data Bank with acquisition code 1OOE.
Results and Discussion.
The molecule of ceDHPR is structurally homologous with mammalian DHPR as shown in Figure 1. After a least-squares fit in O, the root-mean-square deviation (RMSD) between 230 Cα atoms of the rat DHPR (PDB code 1DHR) and ceDHPR is 1.12 Å. Similarly, the RMSD between 229 Cα atoms of the human DHPR (PDB code 1HDR) and ceDHPR is 1.16 Å. Corresponding dimers can also be well superimposed. For instance, the RMSD is 1.33 Å for 450 Cα atoms between the dimers of ceDHPR and rat DHPR. This shows that the dimeric assembly is also conserved in ceDHPR crystal structure. The subunits interact through a four-helix bundle (two helices from each subunit) as observed in mammalian DHPR structures. The mammalian DHPR have been crystallized in space groups C2 and C2221, and the dimer subunits are related either by the noncrystallographic twofold axis or by the crystallographic twofold. In the ceDHPR structure, the two subunits are related by the noncrystallographic twofold.
The relatively high resolution of ceDHPR structure (1.65 Å vs 2.3 Å for 1DHR and 2.5 Å for 1HDR) reveals unambiguously that the noncrystallographic twofold symmetry is only approximate. As a result of different interactions in the crystal packing, local differences between the subunits are found not only in conformations of side chains but also, to a lesser extent, in the conformation of the main chain. The ϕ,ψ angles of the residue Gln69A fall in the unexpected region of the Ramachandran plot,14 although there is good electron density throughout the chain and the real space R-factor value for Gln69A is 5.3%. On the contrary, the residue Gln69B is in a favored region of the Ramachandran plot, supported again by good electron density. In subunit A, the carbonyl group of Gly70A is engaged in a hydrogen bond with the peptide nitrogen of the residue Asp166B of a symmetry-related molecule. Apparently, this causes a flip of the peptide group between Gln69A and Gly70A, which brings the residue Gln69A to an unfavored conformational region. In subunit B, the symmetry-related environment of Gln69B is different and unable to form any hydrogen bond. Therefore, the residue Gln69B is not forced to an unusual conformation by symmetry interactions.
Binding of MES from the crystallization buffer seems to be casual. In both subunits, the molecule of MES is apparently hydrogen bonded through its SO3 group to side chains of the residues Lys207 and Trp208, which are not involved in the active site.
It has been proposed4, 6 that binding of NADH is essential for both retention of DHPR stability and for generation of the active site. Remarkably, the structure of ceDHPR has been crystallized as an apo enzyme, refined at higher resolution, and found structurally homologous to the mammalian DHPR/NADH complexes studied previously. A close inspection of the superimposed structures shows indeed that segments of the main chain in the active site region are slightly shifted (1–2 Å) toward NADH in the liganded structure. However, there are no major conformational changes in side chains of the conserved active site residues, and it appears that the active site is essentially preformed in the unbound state, making a tighter “grip” on the bound NADH molecule. Although about 66% of the NADH surface is buried on binding to DHPR and the qBH2 binding site of ceDHPR is partially occupied by a symmetry-related molecule, it is still conceivable that the molecule of NADH just slips into its binding site, which is the largest crevice on the DHPR surface, and no major conformational changes are necessary in DHPR main chain.
Because there is no crystallographic data on any DHPR/substrate complex, the molecule of qBH2 was modeled in an appropriate distance from C4 atom of the nicotine ring of the bound NADH.4 Although the modeling can explain the hydride transfer from NADH, it also shows that there is no conveniently positioned group for protonation of the substrate. Therefore, the proton is expected to come from the bulk solvent region, and the active site must remain relatively open. The modeling also shows that if there are no major conformational changes in the DHPR main-chain, the N3 atom and 2-NH2 group of qBH2 are solvent accessible. Thus, the functionality of the enzyme is consistent with the concept of a preformed active site observed in the apo structure of ceDHPR.
We thank John H. Wang for his help in protein purification. The diffraction data were collected at beamline 22-ID in the facilities of the South East Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source (APS), Argonne National Laboratory. Supporting institutions may be found at www.ser.anl.gov/new/index.html. Use of the APS was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. W31-109-Eng-38.
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