Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A2 (TXA2) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C4 (LTC4)-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA2 analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA2 analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA2 analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC4 and occupies a very similar position to LTE4 in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from  2 and cysteinyl leukotrienes, two substances involved in blood vessel constriction, platelet aggregation, and inflammatory responses to an insect bite. We produced this protein in bacteria and showed that it tightly binds both these molecules, inhibiting the processes in which they are involved. We then determined its structure using X-ray crystallography and showed that there is a single binding site in one domain of the protein, accommodating both thromboxane A2 and cysteinyl leukotrienes, and that this site is responsible for the scavenging effect of the protein. These studies reveal the structural features of proteins needed to bind to key molecules of potential pharmacological importance and add to our understanding of the process of mosquito blood feeding, which is essential for transmission of the malaria parasite.When feeding, a female mosquito must inhibit the blood clotting and inflammatory responses of the host. To do this, the insect produces salivary proteins that neutralize key host molecules participating in clotting and inflammation. Here, we describe a salivary protein AnSt-D7L1 that scavenges both thomboxane A Hematophagous arthropods produce a varied mix of salivary proteins, peptides, and small molecules aimed at overcoming the hemostatic and inflammatory responses of the host. In order to successfully take a meal, the blood feeder must prevent host vasoconstrictive responses and the clotting of blood as it travels through the mouthparts to the gut 2 biosynthesis and the release of dense granules containing small molecule mediators of platelet activation and inflammation such as ADP and serotonin potentiate the activation response. In sensitized hosts, IgE antibodies recognizing salivary proteins activate mast cells in the skin, leading to the release of large amounts of histamine and the synthesis of CysLTs Wounding as a result of a mosquito bite exposes collagen and other matrix proteins that act to initiate the activation of platelets. The stimulation of TXAAn important functional theme in the physiology of blood feeding is the use of specific salivary binding proteins to sequester small-molecule agonists of inflammation and platelet activation Aedes aegypti, have two OBP domains, with each domain binding a single ligand molecule An. gambiae (referred to as D7r proteins) being extensively characterized and found to bind a single ligand molecule per protein An. stephensi, and one of these has also been found to function as an inhibitor of coagulation and inflammation pathways by binding factor XII(a) and high molecular weight kininogen An. gambiae as the protein D7r1 Anopheles examined, the two-domain long forms of D7 have not been lost, suggesting that they may play, at least in part, a different role in these species.Two protein families have thus far been implicated as scavengers of small molecule modulators of inflammation and platelet aggregation in blood feeding arthropods. The lipocalins serve this function in ticks and triatomine bugs, while members of the D7 family serve the function in mosquitoes An. stephensi, a vector of the malaria parasite. We found that unlike other members of the D7 family, this protein functions as a TXA2-binding protein, thereby acting to inhibit the activation of platelets during feeding. We also describe the crystal structures of ligand complexes that provide, to our knowledge, the first view of the molecular interactions involved in TXA2 binding. We ask whether evolution of the short D7 proteins in Anopheles sp. and their assumption of the biogenic amine binding function otherwise seen in the long form of Aedes has resulted in the evolution of functional changes in the protein. We find that AnSt-D7L1 has acquired the ability to bind the vasoactive platelet aggregation and proinflammatory agonist TXA2 in addition to maintaining the ability to bind CysLTs. Additionally, the C-terminal domain has undergone extensive structural rearrangements resulting in a loss of the biogenic amine binding function.In this study we examine the ligand binding specificity, physiological function, and three dimensional structure of AnSt-D7L1, a two-domain D7 protein from Ae. aegypti  in AnSt-D7L1 and other two-domain D7 proteins from Anopheles species  but bound the prostaglandins PGD2, PGE2, and PGF2α with affinities much lower than those seen with CysLTs and TXA2 analogs  and glypoprotein VI (GPVI) 2 and release of ADP, which act together with collagen to amplify pro-aggregatory signals resulting in “inside out” activation of integrin αIIbβ3, the receptor for fibrinogen and fibrin, and consequent platelet aggregation 2 or ADP 2 and ADP 2 by AnSt-D7L1 should inhibit platelet activation by low concentrations of collagen and have no effect at higher collagen concentrations.Exposure of collagen from the subendothelial matrix at the site of a wound plays a key role in triggering platelet adhesion and activation. Platelet interaction with collagen may be indirect, due to binding of the VWF-collagen complex with glycoprotein Ib-IX-V (GPIb-IX-V) or αWhen we incubated stirred platelets with AnSt-D7L1 and stimulated with a low concentration (1.6 µg/mL) of collagen, we observed a delay in the onset of shape change and a diminished extent of platelet aggregation . The deg2-independent process, however, and should not be limited by an inhibitor targeting TXA2Rhodnius prolixus saliva 2, suggesting that the inhibition of collagen-mediated aggregation by AnSt-D7L1 depends only on its ability to bind TXA2.Platelet aggregation induced by collagen is also affected by apyrase, indicating an important role for ADP. Shape change induced by lower concentrations of ADP is a TXA2 receptor (TP), or were pre-treated with indomethacin, an inhibitor that prevents TXA2 biosynthesis, aggregation was attenuated -induced platelet aggregation in a concentration-dependent manner . At low 12/13 protein signaling cascade, which requires only small amounts of U46619 (EC50 around 13 nM) q/11-coupled signaling Although higher concentrations (2 and 3 µM) of the protein completely prevented aggregation of platelets, shape change was not completely inhibited and could only be prevented by blockade of the TP receptor with the antagonist SQ 29,548 . This is2, as well as of prostaglandins, we tested the effect of AnSt-D7L1 on platelet aggregation induced by this compound. AnSt-D7L1 drastically decreased platelet aggregation induced by AA, showing a similar effect to that observed when platelets were pre-treated with indomethacin  extends away from the surface of the protein and forms a β-turn between Thr 293 and His 296. In the ligand complexes the segment between Leu 291 and Val 295 packs against the surface of the protein, where it participates in a number of intramolecular hydrogen bonds  as well as the C-terminal domain of the two-domain D7 present in Ae. aegypti (AeD7) inhibit inflammation and vascular tone by binding biogenic amines An. stephensi, AnSt-D7L1, binds CysLTs like AeD7 but also functions as an inhibitor of platelet activation by binding TXA2. AeD7 showed no detectable binding with the TXA2 analog U46619, while AnSt-D7L1 binds this ligand with high affinity. Additionally, the C-terminal domain has lost the ability to bind biogenic amines, a function found in apparent orthologs of this protein in culicine mosquitoes.Prevention of host hemostasis and inflammation is the primary function of the salivary secretion of blood feeding arthropods 2 and its analogs contain a hydroxyl group at the ω-5 position , as well as by causing inflammatory skin responses.Unlike the previously described AeD7, AnSt-D7L1 also binds TXA2β1 and αIIbβ3, thereby increasing the affinities for their respective ligands, resulting in aggregation of platelets. Activated platelets release granules containing small molecule agonists such as ADP and begin synthesizing TXA2. These secondary mediators are able to diffuse and activate circulating platelets that do not have contact with collagen, resulting in potentiation of the pro-aggregatory signal and an increase in the size of the platelet plug. Studies comparing the effects of collagen with other GPVI agonists, such as collagen-related peptide (CRP) q have shown that ADP and TXA2 are essential for collagen-induced platelet aggregation.Mosquito bites cause injury to the skin, leading to the exposure of collagen present in subendothelial extracellular matrix 2 generated in response to collagen exposure. Moreover, similar effects have been shown in previous studies where platelets lacking Gαq and Gα13, G proteins coupled to TP and to ADP receptors, were exposed to collagen The effect of AnSt-D7L1 on collagen-induced aggregation is similar to that obtained when platelets are treated with indomethacin or SQ 29,548, strongly suggesting that this protein is able to scavenge the TXA2 or its analogs to TP receptors on the platelet surface activates the platelet, leading to shape change followed by aggregation 2 being produced as a result of stimulation by its biosynthetic precursor. A recently described mutation in the human TP receptor causes platelet dysfunction and lack of responsiveness to AA and U46619 Binding of TXA2 secreted by platelets in response to collagen stimulation and that 0.1 µM eliminates most of the aggregation response  from An. gambiae also show very similar binding modes for biogenic amine ligands, with most hydrogen bonding interactions being highly conserved between the two. Phylogenetic analyses of D7 sequences suggest that the two-domain forms are ancestral and that the single domain proteins found in both culicine and anopheline mosquitoes are derived from an apparent duplication of the C-terminal domain of a two-domain D7 protein The highly expressed single-domain D7 proteins in  binding  The crysSequence comparisons of apparently orthologous two-domain D7s from culicine and anopheline species initially suggested that the C-terminal domain of the anopheline proteins lack some important determinants of biogenic amine binding, while maintaining a high overall degree of similarity with the culicine forms. The work presented here shows that the biogenic amine binding function has indeed been lost in the anopheline proteins and that the loss of function is due to rearrangements that dramatically affect the structure of the binding pocket. The loss of α-helix B2 that is found in both AeD7 and D7r results in Arg 177 taking on a different conformation in AnSt-D7L1. Along with the bulky side chain of Trp 173, Arg 177 fills most of what would be the biogenic amine binding pocket in the D7 forms that bind these ligands . Two cryAedes and Culex) mosquitoes, making it unnecessary for AnSt-D7L1 and other anopheline forms to maintain the biogenic amine-binding function. Apparently, another function has been acquired by the C-terminal domain of these proteins that may be entirely unrelated to the binding of small-molecule ligands. This case seems to be an example of extensive duplication of salivary protein genes allowing structural and functional diversification within protein families. The D7 protein family represents a case where a duplication of one domain of an ancestral two-domain protein to form a one-domain form has allowed the ancestral protein to lose its original function and almost certainly acquire a new one. We are currently working to determine the more recently acquired function of the C-terminal domain in this group of proteins.It appears that the cluster of single-domain D7 proteins in anopheline mosquitoes has taken over the function of the C-terminal domain of the two-domain D7s of culicine , PAF, and SQ 29,548 were obtained from Cayman Chemical. Native collagen fibrils (type I) from equine tendons, AA, and ristocetin used in platelet experiments were purchased from Chrono-Log Corporation. Convulxin and RPAI1 were prepared as described previously UltraPure guanidinium hydrochloride was obtained from Invitrogen. L-arginine, serotonin, norepinephrine, histamine, ADP, PMA, and indomethacin were obtained from Sigma-Aldrich. All leukotrienes and prostaglandins as well as U46619, carboxyclic TXAThe AnSt-D7L1 cDNA, minus its signal sequence, was cloned into the expression vector pET17b and was expressed in BL21(DE3)pLysS cells grown for 3 h after induction with 1 mM IPTG AnSt-D7L1 was prepared for ITC experiments by dialysis against 20 mM Tris-HCl 0.15 M NaCl pH 7.4 for 1 h. Binding experiments were performed on a MicroCal VP-ITC instrument. Aliquots of lipid stock solutions in ethanol or methyl acetate were dried under a stream of nitrogen in a glass vial, then dissolved in 20 mM Tris HCl, 0.15 M NaCl pH 7.4, vortexed, and sonicated for 8 min. Non-lipidic ligands were dissolved in 20 mM Tris HCl, 0.15 M NaCl pH 7.4. All solutions were degassed prior to use. Assays were performed at 35°C with successive 10 µL injections, with a spacing interval of 180 s and a syringe stirring speed of 290 rpm. In all the cases, after subtraction of the heats of dilution, the measured heats were converted to enthalpies and analyzed by fitting to a single-site binding model using the Microcal Origin software.4 was measured isotonically with an initial load of 1.0 g tension, and rat aorta ring contraction in response to U46619 was measured isometrically after a distension of 2.5 g. Tissue preparations were bathed in modified Tyrode solution  during the course of the experiment, and the solutions were kept oxygenated by bubbling air into the bath Guinea pig ileum contraction in response to LTC5/µL final density) and Tyrode-BSA buffer 2 receptor) antagonist SQ 29,548 or made in the presence of proteins (AnSt-D7L1 or RPAI-1), those compounds were added to the platelet suspension and Tyrode mix prior to addition of agonists. In experiments in which indomethacin, a general cycloxygenase inhibitor, was used, platelets were pre-incubated with the compound for 5 min prior to addition of agonists.Platelets were prepared as described previously 4 or U46619 prepared in 10 mM Tris-HCl pH 8.0 buffer was pre-incubated with AnSt-D7L1 for 10 min but in a 1.3 molar excess when compared to the protein concentration. Prior to data collection, the crystals were frozen using the cryoprotectant described above.AnSt-D7L1 was crystallized using the hanging drop vapor diffusion method from 0.1 M Tris pH 8.5, 0.2 M lithium sulfate, 30% PEG 3000. The crystals were frozen for data collection in the crystallization buffer described above, containing 10% glycerol. To prepare a selenomethionine derivative of AnSt-D7L1, the expression plasmid construct was transformed into BL834(DE3)pLysS cells and grown in SelenoMet media (Molecular Dimensions Ltd.) as per the manufacturer's instructions. In order to obtain crystals with the protein bound to its ligands, either LTC12121 and contain one monomer per asymmetric unit , a ligand-free crystal (1.77 Å resolution), a U46619-complexed co-crystal (1.45 Å resolution), and a LTC4-complexed co-crystal (1.43 Å resolution).Data collection was performed at beamlines 22-BM at the Southeast Regional Collaborative Access Team (SER-CAT) and 19-BM of the Structural Biology Center (SBC), Advanced Photon Source (APS), Argonne National Laboratory. Crystals of the ligand-free form of the protein belong to the space group P2ric unit . CrystalAe. aegypti as the search model -hydroxy--7,9,11,14-eicosatetraenoic acid. The structures of ligand-free AnSt-D7L1 (3NGV), its U46619 complex (3NHT), and its LTC4 complex (3NHI) have been deposited with the RCSB protein data bank.The structure of AnSt-D7L1 was determined using a combination of single wavelength anomalous dispersion (SAD) methods with data collected at the selenium absorption edge, and molecular replacement using the N-terminal domain of AeD7 from ch model  [15]. ThFigure S1Structures of leukotrienes and thromboxanes and thromboxane analogs. (A) LTC4, (B) LTD4, (C) LTE4, (D) TXA2, (E) carbocyclic TXA2, (F) U46619, (G) U51605.(1.67 MB TIF)Click here for additional data file.Figure S24 complex (A) and free LTC4 (B) on a Superdex peptide column.Gel filtration of the AnSt-D7L1-LTC (A) AnSt-D7L1 (100 µL) 10 µM and LTC4 10 µM were incubated together to form a complex which was then injected onto the column. (B) LTC4 was injected alone in order to obtain a retention volume measurement for the free ligand. In both cases, the column was eluted with 100 mM ammonium acetate pH 7.2 at a flow rate of 60 µL per min.(0.39 MB TIF)Click here for additional data file.Figure S34 complex after gel filtration chromatography.Mass spectral analysis of the AnSt-D7L1-LTC Top: The peak fraction from the gel filtration of 4 solution. The chromatograms represent abundance of the m/z 626.7 molecular ion (M+H) of LTC4. The inset is a sample collision-induced dissociation spectrum (35% intensity) recorded at 61–65 min.(2.15 MB TIF)Click here for additional data file.Figure S4Structural comparisons of D7 proteins. (A) Stereoview of superposed carbon alpha traces of the C-terminal domains of AnSt-D7L1 (red) and AeD7 (black). Helix B2 of AeD7 and H2 from both proteins are labeled in the appropriate colors. (B) Stereoview of superposed N-terminal domains from ligand-free AnSt-D7L1 and its U46619 complex. The N-termini are labeled. (C) Stereoview of superposed C-terminal domains from ligand-free AnSt-D7L1 and its U46619 complex. The difference in position of the C-terminus for each protein is indicated in the appropriate color.(2.52 MB TIF)Click here for additional data file.Figure S5Purification of recombinant AnSt-D7L1. (A) Gel filtration chromatography of purified AnSt-D7L1 on Superdex 75. Elution buffer: 20 mM Tris HCl pH 8.0, 0.15 M NaCl. (B) SDS-PAGE gel of purified recombinant AnSt-D7L1 stained with Coomassie blue.(5.20 MB TIF)Click here for additional data file.


Trichoderma virens (TPS) is an important member of the NRPS family that exhibits antifungal properties. The majority of the NRPSs terminate peptide synthesis with the thioesterase (TE) domain, which either hydrolyzes the thioester linkage, releasing the free peptic acid, or catalyzes the intramolecular macrocyclization to produce a macrolactone product. TPS is an important NRPS that does not encompass a TE domain, but rather a reductase domain (R domain) to release the mature peptide product reductively with the aid of a NADPH cofactor. However, the catalytic mechanism of the reductase domain has not yet been elucidated.Nonribosomal peptide synthetases (NRPSs) are multienzymatic, multidomain megasynthases involved in the biosynthesis of pharmaceutically important nonribosomal peptides. The peptaibol synthetase from We present here a three-dimensional (3D) model of the reductase domain based on the crystal structure of vestitone reductase (VR). VR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and is responsible for the nicotinamide dinucleotide phosphate (NADPH)-dependent reduction of the substrate into its corresponding secondary alcohol product. The binding sites of the probable linear substrates, alamethicin, trichotoxin, antiamoebin I, chrysopermin C and gramicidin, were identified within the modeled R domain using multiple docking approaches. The docking results of the ligand in the active site of the R domain showed that reductase side chains have a high affinity towards ligand binding, while the thioester oxygen of each substrate forms a hydrogen bond with the OH group of Tyr176 and the thiol group of the substrate is closer to the Glu220. The modeling and docking studies revealed the reaction mechanism of reduction of thioester into a primary alcohol.Peptaibol biosynthesis incorporates a single R domain, which appears to catalyze the four-electron reduction reaction of a peptidyl carrier protein (PCP)-bound peptide to its corresponding primary alcohol. Analysis of R domains present in the non-redundant (nr) database of the NCBI showed that the R domain always resides in the last NRPS module and is involved in either a two or four-electron reduction reaction. Trichoderma virens peptaibol synthetases (TPS) are important NRPS members that synthesize peptaibols, which are a family of short chain length peptides (≤20 residues) that harbor a unique C-terminal alcoholic group instead of a carboxyl group. Peptaibols form right-handed alpha-helical structures that self-associate into multimeric transmembrane channels. These channels conduct ionic species to disrupt the osmotic balance and promote cell death [Nonribosomal peptide synthetases (NRPSs) are multi-enzymatic, multi-domain megasynthases that are involved in the synthesis of a remarkable array of commercially important nonribosomal peptides through the sequential condensation of amino acid monomers -4. Trichll death . Peptaibll death .Peptaibol is synthesized by the TPS assembly line complex, which is a nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) hybrid system. Various hybrid NRPS/PKS systems such as rapamycin, bleomycin, yersiniabactin and epothilone have already been reported in the literature ,8. In TPet al. [During biosynthesis, the growing chain remains covalently attached to the enzyme until it reaches its full length, at which point the TE domain residing at the last module releases a cyclic, branched-cyclic or linear product ,14. An aet al.  already Brevibacillus texasporus (BT) by using BLAST searches of the nr database. The primary sequences of R domains from TPS and glycopeptidolipid show 60% sequence similarity. In order to determine the reduction reaction catalyzed by the R domain, which is common for the above synthase families, we chose glycopeptidolipid having 20.5% sequence identity with VR. Our modeling and refinement approach to explain the reduction reaction mechanism is similar to the approach used by Zhou et al. [1BG4). They subsequently conducted molecular dynamics (MD) refinement and tested the inhibitors targeting the heparanase model. We constructed a 3D model of the R domain based on vestitone reductase, which is a member of the SDR family for which the 3D structure has been solved by X-ray crystallography. The refined structure of the R domain was employed in the docking simulation. Analysis revealed that Tyr176 and Glu220 are the prime proton donors for the reduction of thioester into the primary alcohol via an intermediate aldehyde. Additionally, the reaction mechanism of the R domain was clearly described herein.In this study, we identified the location of the R domain that catalyzes reduction of the PCP-bound peptide to the corresponding primary alcohol in the 422 amino acid C-terminal linker region of TPS Figure . To elucu et al. , who modThe TPS protein sequence extracted from the NCBI protein database (accession no. AAM78457) was submitted to NRPS-PKS, which is a database that predicts the domain organization of nonribosomal peptide synthetases and polyketide synthetases . This prhttp://www.psipred.net/psiform.html[http://cl.sdsc.edu/[http://www.tcoffee.org/ to identify the conserved regions of the motif among these characterized proteins.A search was conducted to identify the possible folds for the R domain (R domain from TPS and similar characterized multidomain proteins containing the R domain) by the threading and fold recognition servers, GenTHREADER form.html and PHYRform.html. We optesdsc.edu/. The cur2p4h chain X) [2p4h and R domain were aligned and carefully checked to avoid deletions or insertions in conserved regions and corrected manually wherever necessary. A series of 10 R domain models were independently constructed with the MOE using the Boltzmann-weighted randomized procedure [The glycopeptidolipid R domain FASTA sequence and the crystal structure coordinates of the VR (chain X)  were loarocedure  combinedrocedure . There wrocedure ,30. The -5 bar-1. A cut off radius of 1 nm was used in the simulation for non-bonded interactions. After equilibrating the system, a 2-nanosecond (ns) production simulation was conducted with a 1 femtosecond (fs) time step at a pressure of 1 bar and a temperature of 300 K.MD simulations were conducted using GROMACS 3.3.3 . The R dThe Site Finder module of MOE 2008.10 was used to identify the possible substrate binding pockets within the newly generated 3D structure of the R domain. Hydrophobic or hydrophilic alpha spheres served as probes denoting zones of tight atom packing. These alpha spheres were used as centroids for the creation of dummy atoms used to define potential sites throughout the docking process ,35.1AMT), antiamoebin I (1GQ0), trichotoxin (1M24), chryospermin C (1EE7) and gramicidin (1NRM) [The reductase substrates included linear peptides such as alamethicin (n (1NRM) -39. The n (1NRM) . Partialn (1NRM) . All of A binding region is identified by a cluster of hydrophobic and hydrophilic alpha spheres; each of which marks their own environments. Ligand atoms are matched to the corresponding alpha spheres during the docking process. The alpha spheres are used to calculate the shape complementarity of small molecules fitting into macromolecules, as well as the binding affinities of the conformers. The above method may generate bound conformations that approach crystallographic resolutions . The ligtotal. For each ligand, 500 conformations were generated using the default systematic search parameters in the ASEDock module. Five thousand poses per conformation were randomly placed onto the alpha spheres located within the large receptor site. From the resulting 500,000 poses, the 200 poses with the lowest Utotal values were selected for further optimization with the MMF94S force field. During the refinement step, the ligand was free to move within the rigid binding pocket.Alpha Sphere based protein-ligand Docking (ASEDock) is a novel fast-docking program written in scientific vector language (SVL) (MOE platform) and based on the alpha shape method. Ligand atoms have alpha spheres within 1 Å. Based on this property, concave models can be created and ligand atoms from a large number of conformations generated by superimposition with these points can be evaluated and scored by maximum overlap with alpha spheres and minimum overlap with the receptor atoms. The ligand conformations were subjected to energy minimization using the MMF94S force field  and when2P4H, devoid of ligand) was retrieved from PDB and the vestitone ligand was downloaded from PubChem for docking. Vestitone-VR docking poses were obtained using MOE-Dock 2008.10 and ASEDock and then compared with the original crystal structure reports.The two docking methods described above were used to assess the validity of the docking predictions based on the R domain-linear substrates  by calculating possible bound conformations of the vestitone-VR. The crystal structure of VR  and a dTDP-glucose 4,6-dehydratase  showed alignment with most R domains. However, considering the alignment length and length of the query sequence, 2p4h was selected as a template for the R domain for further analysis. These results suggest that the R domains present in NRPS proteins are likely to be around 300 amino acids long. In addition, we used 2p4h as the baseline point and superimposed it onto the PDB structures listed in Table The lack of a crystal structure for any R domain from the NRPS biosynthetic pathway and the high degree of sequence divergence in this family prompted us to use threading and fold recognition approaches. The GenTHREADER and PHYRE fold recognition prediction servers were used for threading analysis because they have the potential to reveal structural similarities in the absence of a high degree of similarity among sequences. All of the C-terminal linkers of the characterized proteins with differing lengths that were identified by the NRPS-PKS program were analyzed using both fold recognition servers Table . The fole-value cutoff was less than the standard cutoff. These findings suggest that no other domain flanks the R domain. An earlier report suggested that the chemical structure of gramicidin A shows a four-electron reduction of the respective thioester, and that the second NADPH-dependent dehydrogenase LgrE, which is encoded by 3 kb upstream of the gene, is required to reduce an aldehyde intermediate into a primary alcohol product [Next, we performed a BLAST search for 10 characterized proteins against the nr database to identify multidomain proteins containing the R domain. We eliminated single-domain proteins from the search results because our query was a multidomain protein and the cutoff amino acids length was ≥ 1000. Table  product . However2p4h). Although this identity level is not enough for homology modeling, its high similarity with VR (~44%), as suggested by our threading analysis, enabled us to model the R domain. The secondary structure of the R domain primary sequence predicted by the PHD server is shown in Additional file The R domain sequence has a low sequence identity (~20.5%) with VR, for which the 3D structure is available in the PDB  as shown in Figure The alignment of the primary structure of the R domain along with its template, l Figure . Ten str2p4h produced by the verify 3D structure evaluation server are shown in Figure The final substrate-free model of the R domain was examined for the distribution of Φ and Ψ angles using Ramachandran plots generated within the MOE. The evaluation indicated that only 4 of 320 residues (1.25%) in the R domain are present in the outlying region, and that these residues were located far away from the active site of the model. Therefore, no further refinement of the model was required. The 3D structure profiles of both the R domain and pot) of the R domain is shown in Additional file The modeled reductase structure was refined and assessed for stability using MD simulation. The MD simulation is useful for identification of potential problems that may lead to instability of the modeled structure. After minimization and equilibration (~360 ps), 2-ns MD simulation was performed and data were collected for analysis. The potential energy (E2p4h) and its ligand, vestitone, were downloaded from Pubchem and used to conduct two different docking simulations. The dominating clusters from the MOE-Dock 2008.10 and ASEDock docking simulations were found to have the same binding orientation when compared with the original VR crystal structure reported by Shao et al. [et al. [To evaluate our docking simulation, the crystal structure of VR  with the linear substrates alamethicin, antiamoebin I, trichotoxin, chrysopermin and gramicidin Figure . These fThe pocket near the catalytic site, which is presumably a substrate-binding site, is formed by loop α6-α7, loop β7-β6, helices α6, α7 and α8, and strands β6, β9 and β11. The amino acids that play a pivotal role in substrate recognition and catalysis are Ser145, Thr150, Val151, Val155, Asn170, Tyr171, Glu172, Tyr176, Leu177, Lys180, Asn210, Asp220, Asn221, Tyr224, M241 and D246 Figure . A closeint) for the substrates within the active site were calculated using the AMBER94 force field  onto the R domain, after which we minimized the energy  group. The distance between the C4 atom of the nicotinamide ring and the thioester carbonyl carbon atom of the substrates is shown in Table Finally, we placed a NADPH in the R domain receptor site by superimposing y Figure . NADPH cThe difference in the VR and reductase dockings and the catalytic activities were further evaluated by detailed analysis of the predicted binding sites using Computed Atlas of Surface Topography of proteins (CASTp) analysis ,56. BaseThe sequence-structure alignment plays a major role in providing reasonable and accurate structural predictions. However, in the case of low sequence identity, MOE has been employed to generate reasonable models for unknown 3D structures . BecauseDocking calculations are generally used to predict ligand-protein conformations and help perform virtual screening of compounds present in chemical databases to identify therapeutic lead compounds -60. GeneDespite the different protein sequences and cognate substrate molecules, all ligand docking algorithms were located on the optimal binding pocket for alamethicin, trichotoxin, antiamoebin I, chrysopermin and gramicidin within the R domain model that was superimposable with the vestitone-VR docked structure Figure . The comet al. [12-Trp13-Leu14-Leu15-Gly16-S-CoA and subsequently replacing Gly16 with Leu, Ala, Asp, Phe and Lys. They showed that R domain appears to be unspecific for the substrates.Further, our docking studies clearly indicated that the R domain was not influenced by the substrate amino acid chain. We have used five different substrates, whose last amino acids (L-configuration) of each substrate are Trp, Pro, Val, Phe and ethanolamine (ETA), respectively. Though they have different physiochemical properties, they bind exactly the same orientation with the R domain. Our findings are also supported by the study conducted by Schracke et al. , in whicBecause the substrate lengths were ≈18-20 amino acids, we used the C-terminal end slots of 3, 4, 5 and 6 amino acids (residues) for the docking experiment to determine how many residues from the substrates could be accommodated in the active site of the R domain to catalyze the reaction mechanism. We found that the final 4 residues of the substrates bind in the active site, but that if there are greater than 4 residues the slot overlaps with the NADPH binding site. These results strongly imply that only the final four residues bind in the same catalytic site, while the remaining residues face away from the catalytic site and might be involved in the interaction with the side chain of the modeled R domain.+. The hydrolysis of the C-S bond in the thioacetal forms an aldehyde bound to the Tyr176. Next, the phosphopantetheinyl sulfur (S-) accepts a proton from COOH group of Glu220 present in the active site, which results in the release of a phosphopantetheine SH group, at which point the second round of reduction can take place in the active site. However, before the second round of the reduction reaction, the previous NADP+ will be released and the second NADPH will bind to the same Rossmann fold. During the second round of reduction, Tyr176, which is the proton donor of the carbonyl oxygen of the bound aldehyde, facilitates the second hydride transfer from NADPH, forming and releasing a primary alcohol. During this step, Tyr176 acts as a general base that is critically involved in the catalysis, which is known to be a common function of Tyr among all SDR family members [A BLAST search for R domains and our sequence analysis of primary alcohol products that possess a single R domain, such as myxalamid, lyngbyatoxin and peptaibols ,40,43-48 members . It shou members . Also, s members ,62. Thus2p4h, while an aldehyde-forming R domain (e.g. saframycin) showed only 15% sequence identity with 2p4h. This prevents modeling of the aldehyde-forming R domain because modeling is generally only feasible when the sequence identity is >20% [1r6d was < 17%, which also makes modeling of the aldehyde-forming R domain difficult. These findings indicate that there may be a structural difference between the alcohol- and aldehyde-forming R domains. The only limitation at present is the lack of a suitable template for modeling aldehyde-forming R domains to explore their function. Further studies in this direction are required to gain a complete understanding of the aldehyde-forming R domains.However, a few studies have reported that free aldehyde products such as nostocyclopeptide, lys2 and saframycin are observed soon after the first round of the reduction reaction -51. The  is >20% ,63. AddiThe results of receptor modeling and docking simulations demonstrate that, in NRPS proteins, the R domain always resided at the extreme C-terminal end that catalyzes the reduction of acyl thioester into its primary alcohol via an aldehyde intermediate through the four-electron reduction reaction. This four-electron reduction reaction was clearly described herein. Our study not only provides insight into the unknown TPS reaction mechanism, but also serves a starting point for identifying point mutations in active site residues that can enhance the selectivity and activity of the substrate.BM designed the experiments, conducted the analysis and wrote the manuscript. SKM helped in construction of the manuscript. GL and SC supervised the work. All authors read and approved the manuscriptList of the proteins containing putative R domains. Among the 252 multidomain proteins, 226 proteins (92%) contained putative R domains at their extreme C-termini end, which might be involved in the product termination, while a stretch of 100-120 amino acids was present adjacent to the putative R domain at the C-terminal in the remaining 26 proteins.Click here for fileDomain organization of sequences after R domain. The sequences after R domain were collected and analyzed by CDD. The domain organizations of the entire length sequence are shown.Click here for fileSecondary structure prediction of TPS reductase. The secondary structure was predicted by PHD. Among 320 residues, 101 (31.5%) residues are α helix, 52 (16.5%) are β sheet and 167 (52%) are random coil.Click here for fileThe potential energy plot of 2 ns MD simulation. During the simulation, the potential energy decreases from the start and then goes to a plateau after 1200 ps. The system intends to be stable.Click here for file


N. gonorrhoeae (UniProt Q5F5IO) reveals that the N-terminal domain assigned as a domain of unknown function (DUF2063) is likely to bind DNA and that the protein may be involved in transcriptional regulation.The crystal structure of the NGO1945 gene product from  Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from  It contains an N-terminal DUF2063 domain (residues 10–92) that has recently been classified as a new Pfam family, PF09836  and 14 (including predicted RNA polymerase sigma factor and repair proteins) are from different strains of Haemophilus (40–90% sequence identity). We have determined the crystal structure of NGO1945 to 2.1 Å resolution in order to expand structural coverage of novel protein-sequence space and assign putative functions to novel proteins that have not been previously studied and whose functions cannot be inferred from sequence homology. This study allows us to assign a putative molecular function to the NGO1945 protein and to the DUF2063 domain.NGO1945 from 2.2.1.et al., 2008N. gonorrhoeae FA 1090 genomic DNA using PfuTurbo DNA polymerase (Stratagene) and I-PIPE primers  that included sequences for the predicted 5′ and 3′ ends. The genomic DNA used here contained a single amino-acid substitution, P94L, when compared with the available GenBank sequence from N. gonorrhoeae FA 1090. The expression vector pSpeedET, which encodes an amino-terminal tobacco etch virus (TEV) protease-cleavable expression and purification tag (MGSDKIHHHHHHENLYFQ/G), was PCR-amplified with V-PIPE primers . The V-PIPE and I-PIPE PCR products were mixed to anneal the amplified DNA fragments together. Escherichia coli GeneHogs (Invitrogen) competent cells were transformed with the V-PIPE/I-PIPE mixture and dispensed onto selective LB–agar plates. The cloning junctions were confirmed by DNA sequencing. Expression was performed in selenomethionine-containing medium. At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 µg ml−1 and the cells were harvested and frozen. After one freeze–thaw cycle, the cells were homogenized in lysis buffer  and the lysate was clarified by centrifugation at 32 500g for 30 min. The soluble fraction was passed over nickel-chelating resin  pre-equilibrated with lysis buffer, the resin was washed with wash buffer  and the protein was eluted with elution buffer . The eluate was buffer-exchanged with HEPES crystallization buffer  using a PD-10 column  and incubated with 1 mg TEV protease per 15 mg eluted protein. The protease-treated eluate was passed over nickel-chelating resin  pre-equilibrated with HEPES crystallization buffer and the resin was washed with the same buffer. The flowthrough and wash fractions were combined and concentrated to 16.2 mg ml−1 by centrifugal ultrafiltration (Millipore) for crystallization trials. NGO1945 was crystallized using the nanodroplet vapor-diffusion method  ethanol and 0.1 M imidazole pH 8.36. A rod-shaped crystal of approximate dimensions 20 × 20 × 100 µm was harvested after 34 d at 277 K for data collection. To determine its oligomeric state in solution, NGO1945 was analyzed using a 1 × 30 cm Superdex 200 size-exclusion column  coupled with miniDAWN static light-scattering (SEC/SLS) and Optilab differential refractive-index detectors (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl and 0.02%(w/v) sodium azide. The molecular weight was calculated using ASTRA 5.1.5 software (Wyatt Technology).Clones were generated using the Polymerase Incomplete Primer Extension (PIPE) cloning method  data were collected at SSRL on beamline BL9-2 at wavelengths corresponding to the high-energy remote (λ1) and inflection (λ2) of a selenium MAD experiment. The data sets were collected at 100 K using a MarMosaic 325 CCD detector. The MAD data were integrated and reduced using MOSFLM  data set and excluding diffraction maxima present in ice rings spanning the resolution ranges 3.97–3.82, 3.72–3.61, 3.50–3.39, 2.69–2.64 and 2.26–2.24 Å and an additional 14 unusually strong reflections (near ice rings) that had intensities 15× greater than the average intensity for their resolution bin. The removal of reflections affected by ice rings resulted in a difference in completeness between the λ1 and λ2 data sets. To ensure that the 14 unusually strong reflections were indeed spurious (near ice rings) and did not reflect real intensities from the protein component of the crystal, we calculated structure factors from the final model after refinement, which revealed that these 14 reflections were not unusually strong. This analysis confirmed that the 14 reflections in the measured data should be removed from the refinement data set. Crystallographic data and refinement statistics are summarized in Table 1No additional cryoprotectant was added to the crystal during data collection. Initial screening for diffraction was carried out using the Stanford Automated Mounting system  was adapted from an analysis using PDBsum  or helices H8, H9 and H11 , respectively] with total buried surface areas of 1550 and 1930 Å2 and ΔG               int values of −53.2 and −82.0 kJ mol−1, respectively. Analytical size-exclusion chromatography in combination with static light scattering (SEC/SLS) revealed the oligomeric form in solution to be a dimer, but with medium confidence . An imidazole molecule (likely to be from the buffer) is bound on the surface to Glu114, Asp166 and Arg162, but its significance is known.The crystal structure of NGO1945 was determined to 2.10 Å resolution using the MAD method Fig. 1. Data-coDALI server , 2.9 Å r.m.s.d. over 61 Cα atoms, 0% sequence identity; Fig. 3a] and the σ3 domain of sigma factor σ28 FliA from Aquifex aeolicus . The N-terminal domain is also similar to the ∼70-residue SAM  domain (SCOP fold 47768), which contains a helix–hairpin–helix (HhH) motif , which is involved in interaction with the C-terminal domain (CTD) of the α subunit of RNA polymerase to inhibit RNA binding during transcription termination. It is also seen as the N-terminal domain of STE50, a modulator of mitogen-activated protein kinase signaling in yeast . SAM domains are involved in protein–protein interactions, either for self-association or for binding to non-SAM-domain proteins  and electrostatic surfaces  support the possibility of this domain being involved in protein–protein interactions, as well as DNA/RNA binding, via charged residues in a basic patch (separate from the dimerization interface) that is comprised of Arg36, Arg39, Arg48, Arg54, Lys60, Arg64, Lys66, Arg71 and Arg74. The surface-exposed aromatic residues Tyr34, Phe45, Trp62 and Phe80 in the monomer, which are mostly hidden in an N-terminal dimer, could play a role in base-stacking interactions with DNA if the functional form of the protein requires dissociation into monomers, but this is only speculation at this point. Phe13 and Arg18 in the disordered N-terminus may also become ordered on binding to DNA and are conserved in many PF09836 proteins , suggesting functional importance.The ordered region of the NGO1945 N-terminal domain (residues 31–116) includes the DUF2063 domain  and is similar to many proteins that contain α-helical bundles. Some of the significant hits are with the σ2 domain of RNA polymerase sigma factor SigR from 3.3.b) are similar in structure to the WW domain  of human FE65  and to the intensely sweet protein monellin . Some similarity is also found to the N-terminal domain of the ribosomal protein L11 from Thermotoga maritima . Thus, this region (residues 138–174) with its negatively charged surface  may be involved in interactions with a binding partner.An ∼20-residue loop (residues 117–137) connects the N-terminal domain to the remainder of the protein and could facilitate movement of the N-terminal and C-terminal domains with respect to each other. Residues 138–174  belonging to SCOP fold 46688, examples of which include the Z-DNA-binding domain of the vaccinia virus E3L protein  and the globular DNA-binding domain of the histone protein H5 . Of the E3L residues that are implicated in Z-DNA binding , the only residue that is conserved in NGO1945 is Lys209 . The GH5 residues that are involved in DNA binding include His25, Lys40, Arg42, Lys52, His62, Lys69, Arg73, Lys85 and Arg94, none of which are conserved in NGO1945. Significant similarity also occurs with DNA-binding eukaryotic transcription factors  in SCOP fold 47453 (superfamily 47454), such as the DNA-binding domain of the MafG bZIP transcription factor , the Nanog homeodomain transcription factor  and the γ-domain of DNA translocase FtsK . The Nanog residues that could bind DNA are Lys43, Thr47, Gln50, Asn51 and Met54, whereas those in MafG are Lys53, Arg56, Arg57, Lys60, Asn61, Tyr64, Ala65, Cys68 and Arg69. As in E3L, the DNA-binding residues are on the same DNA recognition helix . None of these Nanog and MafG residues are conserved in NGO1945.Residues 175–231 of NGO1945 are similar to DNA-binding proteins . A β-strand preceding the HTH portion in a wHTH domain is also seen in the DNA-binding domain of the response regulator PhoP . The PhoP residues that may interact with DNA  motif in which the ‘wing’ precedes the helices, as in the transcription repressor MecI , NGO1946 , NGO1947  that has been implicated in invasion and virulence in Legionella pneumophila (Polesky et al., 2001Burkholderia and Bordetella homologs are present with hemolysin-related and exported proteins.The genomic context of other DUF2063 homologs supports an involvement in virulence. For example, MCA3109 and ABO1516, which are DUF2063 homologs from ngo1945 are consistent with a possible role in transcription pathways and indicate that the members of this family may be transcription factors; the genome context further supports involvement in virulence. Alternatively, they may have some novel functionality that remains to be determined. Since significant sequence homologs of NGO1945 are primarily found in different strains of the bacteria Neisseria and Haemophilus, which are human pathogens that are involved in sexually transmitted diseases, in meningitis and in ear, eye or sinus infections in infants and children, further structure-based biochemical investigation of NGO1945 may be of therapeutic value.In conclusion, the NGO1945 crystal structure allows the assignment of a putative function for this protein and for the PF09836 family in general. Structural similarity to transcription factors and presence of a surface-exposed basic patch in the N-terminal DUF2063 domain indicates the possibility of DNA binding. The nonconservation of DNA-binding residues in NGO1945 compared with structurally similar proteins may give rise to a different mode of DNA binding. The multi-domain architecture, potential DNA binding and genome context of et al., 2010http://www.topsan.org/explore?PDBid=3dee.Additional information about NGO1945 is available from TOPSAN (Krishna N. gonorrhoeae, 3dee, r3deesfNGO1945 from             PDB reference:


The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R               cryst of 19.0% and an R               free of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR,  In most organisms, Trx is reduced enzymatically by NADPH via NADPH-dependent thioredoxin reductase  systems are ubiquitous redox regulators that facilitate the reduction of other proteins et al., 2004et al., 1998et al., 1979et al., 2003et al., 2008et al., 1993et al., 2004et al., 2003et al., 2008Plants exhibit a unique thioredoxin system with a complex time-, tissue- and organelle-specific expression pattern of a diverse selection of Trx isozymes that are not found in other organisms . The crystal structure of Escherichia coli NTR (EcNTR) cross-linked to Trx demonstrated that the proposed reaction mechanism was indeed plausible , while only one yeast  NTR2 (HvNTR2), the first structure of a monocot NTR, which moreover falls into a distinct phylogenetic class of NTRs  (Novagen) with an N-terminal His tag MGSSHHHHHHSSGLVPRGSH as described previously  pre-equilibrated with 10 mM imidazole, 0.5 M NaCl and 30 mM Tris–HCl pH 8.0 and eluted with a 0–100% gradient of 400 mM imidazole, 0.5 M NaCl and 30 mM Tris–HCl pH 8.0. Finally, the protein was dialyzed against 10 mM Tris–HCl pH 8.0, the purity was checked by SDS–PAGE and the sample was concentrated on an Amicon Ultra centrifugal filter unit  to an OD280 of 3.96, which corresponds to a concentration of approximately 2.5 mg ml−1. The His6-HvNTR2 solution was used for crystallization experiments without further purification and was not subjected to thrombin cleavage.Recombinant 2.2.w/v) PEG 6000 (Fluka) and 0.1 M citrate buffer pH 4.0 after 4 d of incubation at 295 K. Fine-tuning of crystallization conditions included screening of the PEG concentration, the effect of the PEG molecular weight and use of the Hampton Research Additive Screen. The optimized conditions consisted of 24%(w/v) PEG 400, 2% Jeffamine M-600, 0.1 M citrate buffer pH 3.5, a protein concentration of 5.7 mg ml−1 and an incubation temperature of 298 K. These conditions gave bright yellow crystals with hexagonal morphology within a week. The diameter of the crystals could reach 0.18 mm. The crystals were flash-frozen directly from the drop without using additional cryoprotectants.Initial crystallization screening experiments were carried out using the PEG 6000 Grid Screen (Hampton Research) and the hanging-drop vapour-diffusion method. Drops of 2.0 µl protein solution were mixed with 2.0 µl reservoir solution and equilibrated over a 500 µl reservoir. Yellow needles were detected in 5% and 5–323 (chain B). The numbering refers to the amino-acid sequence of wild-type HvNTR2. The biologically relevant dimer, inferred by analogy to the E. coli NTR system, is formed around the crystallographic twofold axis. The structure was determined at 2.6 Å resolution and refined to an R               cryst of 19.0% and an R               free of 23.8%. One FAD molecule with well defined electron density and B factors (∼40 Å2) comparable to the surrounding protein is present in each subunit. NADPH did not fit the excess electron density in the expected NADPH-binding pocket. Instead, the density fitted reasonably well to a citrate molecule accidentally present from the crystallization conditions . High B factors but continuous main-chain electron density is found in the N-terminus (residues 6–12), the loop between A1 and B3 (residues 33–35), B5 and surrounding loops (residues 96–105), the loop between B10 and B11 (residues 153–158), B12 and surrounding loops (residues 174–196) and B15 and surrounding loops (residues 220–245) . However, the relative orientation of the two domains in HvNTR2 is quite different from their orientation in AtNTR-B and other low-molecular-weight NTRs in the FO conformation , they differ by a minor translation of 1 Å and by a substantial 65.6° rotation about the two β-strands connecting the domains. However, comparing the structure of EcNTR in the FR conformation with the structure of HvNTR2 shows that they differ by a 6.7% closure, a translation of −1.4 Å and a rotation of 49.8°. The smaller rotation of 49.8° compared with 65.6° indicates that HvNTR2 is actually closer to the FR conformation than other crystallized NTRs, which have all been in the FO conformation. Yet, within the group of NTR structures determined in the FO conformation there are minor variations in the relative orientation of the two domains. Superposition with EcNTR requires an 8° inter-domain rotation for both AtNTR-B and Saccharomyces cerevisiae NTR  shows a very similar orientation of loops, α-helices and β-sheets and the aforementioned variation in relative domain orientation. Some major structural differences are observed in two loop regions when the NADPH domains alone are superimposed . The long loop region between strand B9 and B10 contains four additional residues in AtNTR and therefore has a protrusion. This loop has the sequence S/N/P-F-T/V/A-G-S-G/E-E/K/T/D-G/A-N/P/S-G/N-G in dicot NTRs (the four extra residues are missing in Populus trichocarpa), while monocot NTRs of the A/B type have a H/Y-F-S/P/A-G-S-D-T/A sequence  and van der Waals interactions (involving 25 amino-acid residues) contribute to FAD binding. The hydrogen-bonding residues are conserved among the plant NTRs but are not conserved among all NTRs  was used for comparison with the potential NADPH-binding pockets of AtNTR-B and HvNTR2. The residues involved in the binding of NADP+ in EcNTR and potentially in HvNTR2 and AtNTR-B are listed in Supplementary Table 1HvNTR2 and AtNTR-B and only a few conservative substitutions are found when compared with the actual binding pocket of EcNTR.The binding of NADPPDB code tdf was uAtNTR-B crystal structure and the partly occupied NADPH molecule also found in the pocket was in a distorted NADPH-binding mode. The likely binding of a citrate ion in the HvNTR2 NADPH-binding pocket not only occludes NADPH binding but could also be the cause of the observed change in the relative domain orientation, which obstructs any possibility of NADPH binding owing to spatial limitations. Also, the unassigned electron density below the isoalloxazine-ring system in the HvNTR2 structure might influence the twist and closure in the domain structure.A sulfate ion was found in the NADPH-binding pocket in the EcNTR and HvNTR2 NADPH-binding pockets were examined and showed very similar charge distributions, with a large number of positive charges matching the negative charges of the NADPH phosphates. However, the superposition also showed that there is not enough space in the HvNTR2 NADPH pocket to accommodate the ribose moiety of NADP+ owing to the changed orientation of the FAD domain. Thus, if HvNTR2 represents an intermediate between the FO and the FR states, NADPH would have to undergo a considerable conformational change during catalysis. It appears likely that NADPH binds following the conformational change, which would also be in agreement with the previously observed slight decrease in the observed rate constant for the domain reorientation event with increasing NADPH concentration . However, it is also possible that the observed domain orientation only reflects the binding of citrate in the active site and therefore is of no relevance to the reaction mechanism.The overall charge distributions and shapes of the 3.7.EcNTR (PDB code 1tde), AtNTR-B and HvNTR2  interactions are located in the Gly129 and Thr47 area in 3.8.EcNTR and AtNTR-B are centred on the loop between β-strands B9 and B10. This loop contains an arginine residue (Arg130 in EcNTR) that is conserved in plant NTR sequences . The neighbouring Gly129 and Ala237 within its spatial proximity are each involved in one hydrogen bond to Trx. The last two hydrogen bonds engage the active-site amino-acid residues Cys138 and Asp139 . The binding of Trx could be guided by the two variable loops, ensuring binding to the optimal Trx isoform. The loop area is free to interact with Trx as observed for the FO conformation of EcNTR (Fig. 5This patch, which adjoins the variable loops in the NADPH domain, supplies all hydrogen bonds specific for Trx binding besides those in the active site. The same area provides the interactions for anchoring of the NADPH domain to the FAD domain in the FO state in both TR Fig. 5.10-helix has been predicted to bind to Trx (Zhang et al., 2009A third loop found between strand B3 and a short 3HvNTR2 is an intermediate between the FO and the FR conformations, it shows that there is not room for bound NADPH during the domain-rotation step. The conformational change from the FO to the FR state could be part of the mechanism that secures the release of NADP+ from FR.Association of Trx with the FO conformation prior to NADPH binding might help in defining the NADPH-binding site. Our postulation that Trx breaks the inter-domain contacts as the first part of the reaction mechanism implies that the NTR domain rotation only happens, or only happens sufficiently, when Trx is available and would explain why almost all NTRs crystallized to date have been in the FO conformation. If the structure of HvNTR2, 2whd, r2whdsf            PDB reference: 10.1107/S0907444909021817/be5129sup1.pdf            Supplementary material file. DOI:


Work from our lab demonstrates that the copper coordination mode depends on the precise ratio of Cu2+ to protein. At low Cu2+ levels, coordination involves histidine side chains from adjacent octarepeats, whereas at high levels each repeat takes up a single copper ion through interactions with the histidine side chain and neighboring backbone amides. Here we use both octarepeat constructs and recombinant PrP to examine how copper coordination modes are influenced by octarepeat expansion. We find that there is little change in affinity or coordination mode populations for octarepeat domains with up to seven segments (three inserts). However, domains with eight or nine total repeats (four or five inserts) become energetically arrested in the multi-histidine coordination mode, as dictated by higher copper uptake capacity and also by increased binding affinity. We next pooled all published cases of human prion disease resulting from octarepeat expansion and find remarkable agreement between the sudden length-dependent change in copper coordination and onset age. Together, these findings suggest that either loss of PrP copper-dependent function or loss of copper-mediated protection against PrP polymerization makes a significant contribution to early onset prion disease.Insertional mutations leading to expansion of the octarepeat domain of the prion protein (PrP) are directly linked to prion disease. While normal PrP has four PHGGGWGQ octapeptide segments in its flexible N-terminal domain, expanded forms may have up to nine additional octapeptide inserts. The type of prion disease segregates with the degree of expansion. With up to four extra octarepeats, the average onset age is above 60 years, whereas five to nine extra octarepeats results in an average onset age between 30 and 40 years, a difference of almost three decades. In wild-type PrP, the octarepeat domain takes up copper (Cu Prion diseases are neurodegenerative disorders involving the prion protein, a normal component of the central nervous system. An unusual class of inherited mutations giving rise to prion disease involves elongation of the so-called octarepeat domain, near the protein's N-terminus. Research from our lab and others shows that this domain binds the micronutrient copper, an essential element for proper neurological function. We investigated how octarepeat elongation influences copper binding by examining both the molecular features and the binding equilibrium. We find that elongation beyond a specific threshold, which confers profound early onset disease, gives rise to concomitant changes in copper uptake. The remarkable agreement between onset age and altered copper binding points to loss of copper protein function as significant in prion neurodegeneration. Sc (scrapie conformer), a misfolded form of the normal cellular prion protein (PrPC) that is found ubiquitously throughout the central nervous system C is a GPI anchored glycoprotein possessing a largely α-helical C-terminal domain and a flexible N-terminal domain (Prion diseases are infectious neurodegenerative disorders that arise from accumulation of PrPl domain . Within Scin vitro assays show that recombinant protein containing insert mutations forms amyloid fibrils faster than wild-type ScOctarepeat (OR) expansions alter the properties of PrP and its interactions with cellular components. When expressed in various cell lines, PrP with additional repeats displays detergent insolubility, resistance to proteinase K digestion similar to PrPSc typically retains an intact protease-resistant core region, which includes the protein's C-terminal domain, and remains capable of propagating disease Sc substructure. This suggests that either disease propagation and mechanisms underlying prion-mediated neurodegeneration are separable or, alternatively, that disease resulting from octarepeat expansions is distinct from other inherited prion diseases.After treatment with proteinase K, aggregated PrP2+) with an affinity that approximately matches extracellular copper concentrations in the brain C is not yet known, the demonstrated interaction with copper suggests a number of possibilities, including protection against Cu2+ mediated oxidative stress, copper transport and copper dependent cellular signaling 2+ stimulates PrP endocytosis 2+ depends on the ratio of copper to protein , but this intermediate species is relatively minor. The experiment was repeated for all the expanded OR constructs, and the results for component 3 are shown in 2+. In contrast, the 8 OR construct (four inserts) exhibits persistent component 3 coordination that reaches a maximum at approximately 2.0–2.5 equivalents. For the 9 OR construct, the maximum is shifted to yet higher Cu2+ concentration reaching a maximum at 3.0–3.5 equivalents. In addition to changes in the location of the maximum, there is a shift in the amount of Cu2+ bound in the component 3 mode. For 4–7 ORs, the maximum is approximately 1.0 equivalents. However, for 8 and 9 ORs, 1.5–2.0 equivalents bind in the component 3 mode.To determine the relative concentrations of the different binding modes for each construct, we used non-negative least squares (NNLS) fitting to a set of well characterized basis spectra, as previously described 2+, rPrP shows approximately equal populations of component 3 and non-OR coordination, at 1.0 equivalent each. At higher copper concentrations, component 3 coordination decreases, followed by an increase in component 1 coordination. The behavior of rPrP closely parallels that of the 4 OR construct in 2+ since this coordination mode competes with non-OR binding. The titration of rPrP+5OR, shown in 2+, component 3 remains the dominant species. In contrast, component 3 in wild-type at 5.0 equivalents Cu2+ accounts for only a small fraction (approximately 10%) of the total copper bound species. The shift in the persistence of component 3 coordination is shown in Titrations were also performed with full-length wild-type recombinant PrP (rPrP) and rPrP containing five extra OR segments to give nine total (rPrP+5OR). Titrations with full-length protein require accounting of both OR binding and non-OR binding (involving two His residues between the octarepeat domain and the folded C-terminus), as shown in 2+. Decomposition of the resulting EPR spectra reveals the concentration ratios of Cu2+ bound to OR construct vs chelator. By working at low copper concentration, we ensure that OR constructs coordinate exclusively as component 3, and this is further verified by lineshape analysis of the EPR spectra. We performed independent measurements with the chelators pentaglycine and oxidized glutathione (two glutathione tripeptides linked through a disulfide bond). dK values for Cu2+ are known for each chelator and are similar to the previously determined dissociation constant for wild-type component 3 2+ as a 1∶1 complex, which simplifies the determination of equilibrium constants. The results, as a function of OR length, are reported in dK is approximately 10−10 M, consistent with our previous results dK decreases approximately by a factor of 10. There is a systematic difference between oxidized glutathione and pentaglycine, with the latter reporting lower dK values. Considering the more conservative results from oxidized glutathione, the affinity for Cu2+ with component 3 coordination is at least tenfold higher in expanded OR domains with eight or more repeat units.Component 3 dissociation constants were measured using a competition assay developed by our lab Insertions of extra repeats in humans causes prion disease but the course of disease depends on the specific length of the OR domain. Analyses of case studies find consistently that individuals with insertions of five or more ORs often develop symptoms in their 30 s, approximately three decades younger than most instances of sporadic or inherited prion disease To examine these data more closely, we plotted the age of onset for each individual case against the number of OR insertions in These results were further supported by an analysis via regression trees and cubic regression see . ClassifCART was also employed to find an optimal onset-age separator between the group of low (1–4) and high (5–9) number of OR inserts . It is possible that PrP with more than eight OR inserts exerts a rapid rate of neurodegeneration, perhaps due to yet further alterations in copper binding, that cannot be overcome even in youthful individuals.Disease duration and OR number are also related in a manner that is significant both statistically and clinically/biologically. In our analysis, duration rose almost monotonically from a mean of 0.4 years for one OR insert to a mean of 10.9 years for seven inserts, and then fell to a mean of 2.3 years for nine inserts  as the outcome; comparison of all pairs of means with multiplicity adjustment supported both the rise and fall just mentioned see ; p-value2+ binding properties. As developed above, the longest OR expansions favor component 3 coordination and resist component 1. Thus, component 1 coordination serves as a convenient measure of altered Cu2+ binding properties. 2+. However, at eight and nine ORs , the population of component 1 coordination drops precipitously. For example, at 3.0 equivalents Cu2+, component 1 coordination is nearly 100% for three inserts and drops to approximately 25% for five inserts. Experimental challenges with solid phase synthesis and protein expression prohibited the study of yet longer OR sequences in either polypeptide constructs or full-length protein, respectively; however, the trend to favor component 3 for long inserts is clear and would not reverse for six OR inserts and beyond. Thus, Cu2+ coordination shows a transition between four and five inserts, coincident with the OR length that correlates with early onset prion disease.The most striking relationship with OR insert number we identified is the age of onset, which shows a sudden drop between four and five inserts from 64.4 years to 37.9 years. 2+ in the component 3 binding mode, as observed. However, eight total repeats allows for two equivalents of component 3 coordination. Thus, higher copper levels are required to drive the transition to component 1 coordination. These arguments based on the stoichiometric ratio of OR segments to copper are straightforward. However, an unexpected finding is that expanded OR domains with more than eight repeats exhibit an approximate 10-fold increase in Cu2+ binding affinity. This affinity shift, in concert with His side chain counts favoring two equivalents of component 3, contributes to the decrease in component 1 coordination for OR domains with four or more inserts beyond wild-type.The wild-type OR domain with four repeats responds to increasing copper concentrations by transitioning from component 3 to component 1 coordination. Our data show that this process is preserved in longer OR domains up to seven total PHGGGWGQ repeats. However, for eight repeat segments (four inserts beyond wild-type) and beyond, this transition is significantly inhibited. The biophysical basis for this likely arises from the number of repeats required for component 3 coordination. As demonstrated in our previous work, component 3 involves coordination of approximately four His side chains from adjacent octarepeat segments. If four repeats are required, then OR domains of up to seven total repeats may only take up a single Cu2+, we performed structure calculations using distance restraints tethering four adjacent repeat His side chains to a single copper ion. We examined PrP with eight repeats and two copper equivalents. The C-terminal domain coordinates are from NMR studies. Other than Cu2+-imidazole distances, the OR domain was left unrestrained during energy minimization. The resulting structure is shown in 2+ are omitted and approximately 40 residues on the N-terminal side of the first repeat are not shown.) His imidazoles arrange with an approximate tetrahedral geometry around each copper center. As expected, the expanded OR domain readily takes up two Cu2+ with a relaxed backbone conformation. Also, with eight total repeat segments, the OR domain comprises a significant fraction of the total protein. Although each copper center carries a divalent positive charge, the rest of the 64 amino acids within the expanded OR domain are uncharged and thus comprise a significant hydrophobic domain. PrP with OR inserts show a strong propensity to form aggregates and amyloid. The enhanced hydrophobicity of the N-terminal domain may facilitate interactions between PrPC copies, thus promoting the amyloid assembly process.To gain insight into the three-dimensional characteristics of PrP with an expanded OR domain interacting with Cu2+ was added in proportion to the number of repeats in each chimera, the lag time decreased in the 4 OR construct but increased in the 8 OR construct. Leliveld et al. examined glutathione-S transferase (GST) fusion proteins onto which OR domains of varying length were grafted to the protein C-terminus Sc. Copper promoted multimerization in both short and long OR constructs, but the longer OR domains also exhibited irreversible aggregation in the absence of copper. In contrast, new studies of expanded mouse PrP suggest that OR inserts actually decrease amyloid production To explore the effect of OR inserts on amyloid formation, Dong et al. developed a chimeric Sup35 yeast protein, in which PrP octarepeats replaced the endogenous repeat sequences Sc deposits in the cerebral cortex and associated dementia The OR length also correlates with the progression of prion disease. Expansions of up to four additional repeats gives the phenotype of familial Creutzfeldt-Jakob disease (fCJD), characterized by PrPProgressive elongation of the OR domain leads to alterations of PrP's molecular properties, with influence on the tendency to aggregate, but there must be an additional mechanism responsible for the sudden and profound shift in age of onset observed between four and six inserts. As derived from data in 2+ oxidation state + and Cu2+, contributing to the production of reactive oxygen species. Copper concentrations vary significantly in the synaptic space, a region of high PrP expression. At rest, synaptic copper concentrations are approximately 3.0 uM 2+ in the component 1 mode. Thus, redox protection emerges at high copper levels. For PrP with more than four inserts, transition to component 1 coordination is inhibited, as indicated by the data in These findings point to altered copper binding in lowering the onset age for prion disease. We consider three possible causes. First, loss of component 1 coordination may lead to enhanced redox stress. As analyzed in our papers and elsewhere, component 1 coordination stabilizes copper in the CuC to interact with binding partners on the cell surface. Transgenic mice with alterations of the intervening sequence between the PrP OR domain and the folded C-terminus show significant neuronal degeneration C interacts in a bivalent fashion with a receptor that plays a role involving cellular signaling or regulation. A possible binding partner candidate is the low-density liproprotein receptor related-protein 1, LRP1 C endocytosis, and either elimination of the OR domain or elongation to 14 total repeats completely halts PrPC cycling. These findings point to a copper-dependent conformational change in the OR domain consistent with the component 3 to component 1 transition.Another possibility is that an expanded OR domain interferes with the ability of PrPC to PrPSc. Using protocols for producing synthetic prions from recombinant mouse PrP, Bocharova et al. showed that Cu2+ increases the lag time for conversion to amyloid C with eight or more repeats resists component 1 coordination and is therefore more susceptible than wild-type to misfolding as amyloid. This is certainly consistent with the widespread amyloid observed in GSS resulting from repeat inserts.The third possibility considers the role of copper in the conversion of PrPC copper protein function or loss of copper-mediated protection against conversion to PrPSc.In summary, our findings demonstrate a very strong relationship between changes in copper binding properties and early onset prion disease. The role of the octarepeats in prion disease is enigmatic. Although the OR domain is not part of the protease resistant scrapie particle, it nevertheless modulates disease progression. The studies here point to contributing factors in prion neurodegeneration, and suggest either loss of PrPAll peptides were synthesized using fluorenylmethoxycarbonyl (Fmoc) methods, as previously described E. coli BL21 Star (DE3) cells (Invitrogen), as previous described Syrian Hamster PrP (rPrP(23–231)) was expressed in the pET101 vector (Invitrogen) in All samples were prepared with buffer containing 37.5 mM MOPS and 18.75% glycerol (v/v), as a cryoprotectant, with pH adjusted to 7.4 8GGGTH. The first four histidine residues were linked to the first copper ion and the second copper ion was bound to the next four histidine residues. Calculations were performed that maintained fixed peptide bond distances and angles. Upper and lower limit distance restraints between the copper atom and the histidine residues were used to calculate the model structure. 100 structures were calculated and the lowest energy conformer was retained. The model was then linked to PDB coordinates from PrP(97–231) to create the full-length model structure Calculations for two equivalents of copper loaded into eight octarepeats as component 3 were calculated using the CYANA torsional dynamics program http://www.r-project.org). Complementary analyses, arriving at similar conclusions using nonparametric regression, the bootstrap, and Bayesian change-point modeling, are available in the online For a formal analysis of the relationship between the number of octarepeat inserts and the onset age, we used a standard one-way ANOVA model in which the independent variable corresponded to the number of repeats  and the dependent variable corresponded to the onset age. To account for multiplicities, multiple comparisons were performed using Tukey's adjustment Protocol S1Statistical analysis using nonlinear regression and Bayesian change point models. These findings provide additional support for the relationship between OR length and onset age, and also reveal evidence of shortened disease duration in cases of eight or more inserts.(0.20 MB PDF)Click here for additional data file.


UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK  family.We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements.The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.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 This article can also be viewed as an  O- substituted terminal monosaccharides with a nine-carbon backbone highly expressed on eukaryotic cell surfaces Sialic acids are N- or Gne is the only known gene in the entire human genome that encodes a ROK domain-containing protein.The first two steps of the biosynthesis of Neu5Ac from UDP-GlcNAc are catalyzed by the bi-functional enzyme UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE). GNE contains an N-terminal epimerase domain and a C-terminal kinase domain gne gene. The mutations are located at either the epimerase domain or the kinase domain Three protein isoforms have been described for human GNE, where isoform 1 is ubiquitously expressed and is believed to be responsible for the basic supply of sialic acids. Isoforms 2 and 3 are generated by alternative splicing and show tissue specific expression patterns. Isoforms 2 and 3 have reduced epimerase activities but almost intact kinase activities and may fine-tune the production of sialic acids The overall structure adopts a typical bi-lobal kinase architecture. Both the N-lobe and the C-lobe have the α/β fold. Each lobe consists of a central β-sheet flanked by α-helices on both sides of the sheet. The last helix C-terminal to the C-lobe is part of the N-lobe and perpendicular to the interfacial helix of the C-lobe. Residues 475–498 of the N-lobe are invisible in the electron density map .9–11CxCGx2G(C/H)xE, which forms an HC3 type zinc-finger with residues H569, C579, C581, C586. The zinc-binding motif is a characteristic feature for all ROK family members The GNE kinase domain contains a type I zinc-binding motif GHxPrevious deletion mutations study has suggested that the GNE kinase domain is responsible for dimerization, while a segment of residues between the epimerase and kinase domains, residues 360–382, is a potential site for trimerization 121 with three molecules in the asymmetric unit. Protein interface and assembly analysis using the PISA server 2 per molecule  in the asymmetric unit . In thisE. coli putative ManNAc kinase (PDB 2aa4) was the top hit with twist-adjusted r.m.s. deviation  of 1.94 Å. The structure of a putative sugar kinase from Bacillus subtilis was the second best hit (PDB 1xc3). The other two homologous structures were transcription repressors that belong to the ROK family (PDB: 1z05 and 1z6r). Vibrio cholerae transcriptional regulator (PDB: 1z05) is a homolog of the E. coli Mlc protein (PDB: 1z6r). The latter is a transcriptional repressor that controls the expression of malT, the central transcription activator of the E. coli maltose system E. coli Mlc structure: the N-lobe of GNE kinase domain aligns to the E-domain of Mlc and the C-lobe aligns to the O-domain of Mlc. It is interesting to note that the Mlc O-domain is responsible for the oligomerization of Mlc protein Structural homology search of the GNE kinase domain using the FATCAT  server E. coli glucokinase complexed with glucose (PDB: 1sz2) To evaluate the putative sugar binding site, the sequence of the GNE kinase domain was aligned with that of gne mutations and HIBM Since the identification of the relationship between The first group of residues I557, G559, V572, and G576 is located at the dimerization interface of the C-lobe and mutation of these residues may interfere with dimerization of the kinase domain. It is noteworthy that kinase domain dimerization does not affect the solvent accessibility of G576 , indicatThe location of this group of residues is also close to the residues involved in sugar substrate binding . ResidueThe second group of residues includes those located at the interfacial helices between the N-lobe and the C-lobe, i.e. N519, A524, F528, G708, and M712. Since the interlobar cleft is the site of ATP and carbohydrate binding as well as where phosphoryl transfer occurs, mutation of these residues could change the interlobar movement during catalysis and thus affect the kinase activity of the protein. For example, the first identified HIBM-related mutation, M712T The third group currently includes residue P511. P511 has the highest relative solvent accessibility (>40%) among the 23 mutation sites and is located on a loop region of the structure. The underlying mechanism for the association of P511H and P511L mutations with HIBM is elusive without further data, but mutation of a proline to any other residue type will inevitably change the flexibility of the loop region around this residue and could thus change the allostery of full length GNE in the higher-order oligomeric state.The fourth group of residues includes all the rest of mutation sites in We show here the 3D structure of the N-acetylmannosamine kinase domain of GNE, the only ROK family kinase encoded in the human genome. The kinase domain dimerizes through an interface at the C-lobe. This is consistent with mutagenesis data from other groups on the full length GNE protein E. coli and synthesized commercially  The DNA fragment encoding GNE residues 406–720 was PCR amplified and subcloned into the pET28-MHL vector (gi:134105571) using an In-Fusion dry-down PCR cloning kit (ClonTech). Protein was overexpressed in E. coli BL21(DE3) CodonPlus-RIL cells (Stratagene) grown in terrific broth medium. The culture was grown at 37°C in a LEX bubbling system (Harbinger Biotech. & Engineering Corp.) until OD600 reached 3.0. The temperature of the culture was then lowered to 15°C and the cells were induced with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside and allowed to grow further overnight. Cells were harvested by centrifugation and flash frozen in liquid nitrogen and stored at −80°C until purification. Frozen cells were thawed and resuspended in 10 mM HEPES buffer (pH 7.5) containing 500 mM sodium chloride, 5% glycerol, 2 mM β-mercaptoethanol, and supplemented with 5 mM imidazole, and mechanically lysed using a microfluidizer  at 1,000 bar pressure. The lysate was clarified by centrifugation. GNE protein was bound with nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) at a ratio of 2.5 mL 50% Ni-NTA flurry per litre of cell culture. The bound protein was washed twice with the same HEPES buffer containing 30 mM or 75 mM imidazole, and finally eluted with the HEPES buffer supplemented with 300 mM imidazole. The elutant containing the GNE protein was further purified by Supderdex-75 size exclusion chromatography . The eluted fractions were pooled, concentrated to a final concentration of 40 mg per mL, and stored in a buffer containing 10 mM HEPES, pH 7.5, 500 mM sodium chloride, 5% glycerol and 5 mM dithiothreitol. The purity of the protein was better than 95% judging from SDS-PAGE gel.The cDNA template encoding the kinase domain of GNE was codon optimized for overexpression in Selenomethionine (SeMet) labelling of the protein was carried out using prepacked M9 SeMet growth media kit (Medicilon) following manufacturer's instructions.The ligand-free form crystals were grown at room temperature in sitting drops. A final concentration of 5 mM ADP, 1∶100 chymotrypsin (w/w) were added into the protein stock solution and 0.5 µL protein solution was mixed immediately with 0.5 µL well solution containing 15% polyethylene glycol (PEG) 4000, 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 5.6 and set up for vapour diffusion crystallization. The SeMet crystal used for structure determination was grown in 14.55% PEG4000, 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 6.0, with 1∶100 chymotrypsin (w/w) and 5 mM ADP in a sitting drop setup. Crystals grew to a mountable size within 24 hours. Paratone oil was used to cryo-protect the crystals.Diffraction data of a selenomethionyl derivative of the GNE kinase domain were collected at beamline 19ID of the Advanced Photon Source (APS) at a wavelength of 0.9792 Å. Initial phases were obtained by single wavelength anomalous diffraction with SOLVE and density modification with RESOLVE Datapack S1http://www.molsoft.com/icm_browser.html.Standalone iSee datapack - contains the enhanced version of this article for use offline. This file can be opened using free software available for download at (ICB)Click here for additional data file.Text S1Instructions for installation and use of the required web plugin (to access the online enhanced version of this article).(PDF)Click here for additional data file.