The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein–ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community

Structural Basis for Substrate Specificity in Human Monomeric Carbonyl Reductases

Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Comparisons between MAGE-G1 and MAGE-A3/A4.

<p><b>(A)</b> Comparison of the relative conformations of WH1 and WH2 in the MAGE-G1 structure (open form) and in the MAGE-A3/A4 structures (closed form, shown as semi-transparent cartoons), the original connectivity is shown on the left and the re-refined on the right hand side. <b>(B)</b> Superposition of the MAGE-A3 (orange), MAGE-A4 (green) and MAGE-G1 (pink) structures on the basis of the individual WH1 and WH2 domains.</p

Refinement statistics for the original and re-refined MAGE-G1 NSE-1 structure.

<p>Refinement statistics for the original and re-refined MAGE-G1 NSE-1 structure.</p

Re-analysis of the MAGE-G1 NSE1 model.

<p><b>(A)</b> View of the original (red ribbon) and alternate (black ribbon) choices around the crystallographic symmetry axis (shown as green lines). A single NSE-1 is shown as grey spheres. <b>(B)</b> Electron density maps in the region connecting WH1 and WH2. The 2F_o-1F_c (blue) and F_o-F_c (green) electron density maps (calculated with all atoms between 161 and 171 omitted from the model) are shown contoured at 0.9 σ and 2.4 σ respectively with the domains coloured as for panel A. <b>(C)</b> Comparison of the interfaces between the original (semi-transparent cartoon) and alternative (opaque cartoon) MAGE-G1 models and NSE-1 (shown in the surface representation). The insert shows a detailed view of the additional interface in the alternate model with interacting residues labelled and shown in the stick format and polar contacts shown as dashed lines. <b>(D)</b> Possible MAGE-G1 NSE-1 hetero-tetramer found in the MAGE-G1 NSE-1 crystallographic asymmetric unit with the two MAGE-G1 monomers (shown in green and blue) topologically interlinked.</p

Analysis of MAGE-A3 in solution.

<p>(A-B) Analytical ultracentrifugation of MAGE-A3 constructs with (A) and without (B) the C-terminal peptide required to form type A dimers. The raw absorbance data plotted as a function of radius and time is shown in the top panel, the centre panel shows the distribution of residuals from the fit of the diffusion deconvoluted continuous distribution c(s) model, and the bottom panel shows the distribution of sedimentation coefficient values from the data fit. (C) Small angle X-ray scattering curves for MAGE-A3 (construct 104–314, containing the C-terminal peptide), collected at three different protein concentrations show significant concentration dependence in the Guinier region. (D) Distance distribution function P(r), calculated from the MAGE-A3 SAXS data, the main plot shows the fit of the P(r) function to the data with the distribution in the insert. (E-G) Comparisons of the experimental SAXS data and theoretical SAXS curves calculated from the MAGE-A3 monomer (E), dimer A (F) and dimer B (G). The main plot shows the fit in reciprocal space using the program CRYSOL[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148762#pone.0148762.ref033" target="_blank">33</a>], and the insert shows the fit in real space calculated with the program SCATTER (<a href="http://www.biosis.net/" target="_blank">www.biosis.net</a>).</p

Structures of the two possible dimers present in the MAGE-A3 crystals.

<p><b>(A)</b> Type A dimers linked by insertion of the C-terminal peptide into the cleft between WH1 and WH2. <b>(B)</b> Type B dimers linked by the extended β-hairpin, the insert shows a detailed view of the interface with interacting residues labelled and polar contacts shown as dashed lines.</p