Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available.

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. 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. 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. A stereo ribbon drawing shows the ceDHPR apoprotein in different colors along its sequence, from blue at the N-terminal to magenta at the C-terminal. The human DHPR complexed with NADH has been superposed on the basis of the corresponding α-carbons. It is displayed as a white ribbon with an atomic model of the NADH colored by atom type, with carbons in white. 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.

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.

We report here the crystal structure of the minimal ligand‐binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen‐binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE‐variant or the DEv‐IgG fold. This subgroup includes the ligand‐binding domain of the collagen‐binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen‐binding S.aureus MSCRAMMs identified so far would also contain the same DEv‐IgG fold. A systematic docking search using the C‐terminal region of the fibrinogen γ‐chain as a probe suggested that a hydrophobic pocket formed between the two DEv‐IgG domains of the clumping factor as the ligand‐binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues 408AGDV411 of the γ‐chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.

FKBP12.6 is a novel isoform of FKBP12, which selectively binds to the cardiac ryanodine receptor (RyR2). The crystal structure of FKBP12.6 in complex with rapamycin has now been determined at 2.0 A resolution. The structures of FKBP12.6 and FKBP12 are nearly identical, except for a displacement observed in the helical region of FKBP12.6 toward the hydrophobic pocket. This displacement was not predicted by homology modeling studies. Analyses of the residues that are likely to confer the RyR2-binding specificity are presented.

Rab proteins are small Ras-like GTPases which play important roles in regulating intracellular vesicle trafficking. The nucleotide-binding domain of Rab6 from the malaria parasite Plasmodium falciparum was crystallized with GDP bound to the active site. The MAD phasing technique was used to determine the crystal structure to 2.3 A resolution. Comparisons of the structure of GDP-bound PfRab6 with the recently determined structures of Rab3A in complex with either a GTP analog or with GTP and Rabphillin present structural evidence supporting the traditional model for the molecular GTP/GDP switch in Rab proteins. PfRab6 residues homologous to those distinguishing human Rab6 isoforms, which differ in binding to Rabkinesin-6 in human cells, are located next to the recognized complementarity-determining region (CDR) and constitute a conceptual broadening of that domain. Despite significant observable differences in Golgi ultrastructure, the Rab6 core structure and switch mechanism appear highly conserved when compared with murine Rab3a structures. A significant difference between the PfRab6 and higher eukaryotic Rabs may be the lack of CDR features that allow binding interactions with Rabkinesin-type effectors.