Recently, there has been growing interest in the function and physiological importance of human TANGO2 (transport and Golgi organization 2 homolog), particularly whether it acts as a heme-trafficking protein. To address this question, we experimentally determined the three-dimensional structure of TANGO2. Our crystallographic analysis indicates that interactions between heme and TANGO2 are nonspecific. Structural comparison of the TANGO2 crystal structure with known cysteine Ntn-hydrolases allowed us to identify a putative active site, catalytic residues and a substrate-binding cavity that correspond to residues that are mutated in pathogenic TANGO2 variants. Based on these features, we propose that TANGO2 may utilize fatty-acid derivatives as substrates, suggesting a potential role in lipid metabolism. Mutations in the human TANGO2 gene cause TANGO2 deficiency disorder, a multisystem, life-threatening disease with onset in early childhood. Together, our results provide new insights into the molecular function of TANGO2 and help to resolve the ongoing debate regarding whether it functions as a heme-trafficking protein.

Enzymes containing tungsten are prevalent in the human gut microbiome and play an important role in maintaining overall gut health. A recent study of Eubacterium limosum revealed a new tungsten binding protein Tub that has nanomolar affinity for tungstate and contains a single TOBE domain found in molybdate storage proteins. A full paper about the protein structure has been published recently [Shao N, et. al, mBio, (2024)]. This poster describes the crystallographic aspects of structure solution and structural details.To better understand Tug-tungstate binding and its structural similarity to molybdate storage proteins, high resolutioncrystal structures of Tug in its apo-form and three (5, 7 and 8) tungstate (WO4--) bound complexes were determined using an in-house Rigaku XtalLAB system. Similar to the molybdate binding proteins the 7 kD Tub protein monomer assembles as a hexamer consisting of a trimer composed of three Tub dimers.The hexamer binds eight tungstate oxyanions located at inter-subunit interfaces. Two distinct tungstate binding sites were observed. Two, type-1 binding sites located at the top and bottom of the hexamer along the hexamer's non-crystallographic 3-fold axis and six, type-2 binding sites located on the dimer's 2-fold axis with each monomer containing a single site.A comparison of the Tub type-1 and type-2 binding sites with those observed in other molybdenum binding proteins (ModG and MopII) showed that the two Tub type 1 binding sites were analogous to the type-1 sites found in ModG(PDB entry 1H9K) while the six Tub type-2 binding sites were similar to those found in MopII (PDB entry 1H9R).The type-1 and type-2 sites were found to be linked by a hydrogen bond network (including a water molecule and the side chain of Asn 22) and are assessable via internal positively charged tunnels leading from the molecule's surface to the tungstate binding sites, whose surface entrance closes upon tungstate binding.This work was funded by Bi-Cheng Wang's endowment fund for crystallographic studies, by John P. Rose's NIH (OD 021762) shared instrument grant for the UGA's Rigaku XtalLAB, and by Mike W.W. Adams' NIH (GM 136885) for the biochemical analysis.

Enzymes containing tungsten rather than the ubiquitous and analogous element molybdenum are prevalent in the human gut microbiome, especifically in microbes that contribute to overall gut health. Eubacterium limosum is a dominant human gut organism whose production of beneficial short-chain fatty acids (SCFAs) from lactate involves tungstoenzymes. Here, we characterized E. limosum Tub, a tungsten storage protein. Tub has a sub-nanomolar affinity for tungstate and contains a single TOBE domain first characterized in a molybdate storage protein. Crystal structures revealed Tub assembles as a hexamer composed of a trimer of dimers, capable of binding eight tungstate oxyanions at two distinct binding sites located at inter-subunit interfaces. Tungstate-saturated Tub exhibited unusually high thermal and chemical stability. Glucose-grown E. limosum accumulates tungsten in Tub and has low levels of two tungstoenzymes, termed WOR1 and FDH, which oxidize aldehydes and formate, respectively. Lactate-grown cells contain high concentrations of these two tungstoenzymes where WOR1 and FDH are involved in converting lactate to SCFAs. Glucose-grown cells appear to accumulate tungstate in Tub in preparation for lactate availability in the human gut. Tub and other TOBE-containing proteins are widespread in the human gut microbiome, and gene co-occurrence analysis predicts that there are comparable numbers of TOBE-containing proteins involved in the storage of tungstate as there are that bind molybdate. The results with E. limosum represent an important step for understanding tungsten storage mechanisms for tungstoenzymes within human gut microbes in general.IMPORTANCETungsten metabolism was found to be prevalent in the human gut microbiome, which is involved in the detoxification of food and antimicrobial aldehydes, as well as in the production of beneficial SCFAs. In this study, we characterized a protein in the human gut microbe, Eubacterium limosum, that stores tungstate in preparation for its use in enzymes involved in SCFA generation. This revealed several families of tungstate binding proteins that are also involved in tungstate transport and tungstate-dependent regulation and are widely distributed in the human gut microbiome. Elucidating how tungsten is stored and transported in the human gut microbes contributes to our understanding of the human gut microbiome and its impact on human health.

Plant cell walls are largely composed of polysaccharide polymers, including cellulose, hemicelluloses (xyloglucan, xylan, mannan, and mixed-linkage β-1,3/1,4-glucan), and pectins. Among these cell wall polysaccharides, xyloglucan, xylan, mannan, and pectins are often O-acetylated, and polysaccharide O-acetylation plays important roles in cell wall assembly and disease resistance. Genetic and biochemical analyses have implicated the involvement of three groups of proteins in plant cell wall polysaccharide O-acetylation: trichome birefringence-like (TBL)/domain of unknown function 231 (DUF231), reduced wall acetylation (RWA), and altered xyloglucan 9 (AXY9). Although the exact roles of RWAs and AXY9 are yet to be identified, members of the TBL/DUF231 family have been found to be O-acetyltransferases responsible for the O-acetylation of xyloglucan, xylan, mannan, and pectins. Here, we provide a comprehensive overview of the occurrence of O-acetylated cell wall polysaccharides, the biochemical properties, structural features, and evolution of cell wall polysaccharide O-acetyltransferases, and the potential biotechnological applications of manipulations of cell wall polysaccharide acetylation. Further in-depth studies of the biochemical mechanisms of cell wall polysaccharide O-acetylation will not only enrich our understanding of cell wall biology, but also have important implications in engineering plants with increased disease resistance and reduced recalcitrance for biofuel production.

A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Crystals of bovine trypsin (UniProt ID P00760) is used by SER-CAT as a quality control standard for determining beamline optimization and performance.However, a selenomethionine labeled standard for MAD/SAD energy optimization and other studies was also needed.To provide this MAD/SAD standard a S195A mutant of bovine trypsin with selenomethione labeling was expressed, purified and crystalized.Crystals of the recombinant protein had a similar habit and diffraction quality compared to native crystals.An 18 sec data set to 1.5 Å resolution was collected on beamline 22ID at the selenium absorption edge using a Rayonix MX300HS 10 Hz CCD detector.The data set (99.1% complete) consisting of 180 one-degree images each exposed for 0.1 seconds was collected.Following SER-CAT protocols the data were auto processed using cmdKylin.Phases were then generated using phenix.autosoland phenix.autobuildplaced 190 of the 223 amino acids giving R and Rfree values of 0.2122 and 0.2332, respectively.Refinement (3 rounds) of the auobuilt model was carried out using phenix.refineand converged to give R and Rfree values of 0.1994 and 0.2187, respectively with good stereochemistry.However, inspection of the refined model (COOT) showed that the electron density of three surface loops (totaling 27 residues) was missing with the loss of the C191 -C220 disulfide.The presentation will provide details of the production of selenomethione labeled protein, its crystallization, data collection and structure solution.It will also explore possible causes for the missing loop density.

Diffraction from the soft X-ray region (wavelength > 1.5Å) has played an important role in recent success for macromolecular structure determinations by Native-SAD phasing (reviewed by Rose, Wang & Weiss, 2015; Rose & Wang, 2016).Other unique applications of soft X-rays for structural biology research, which may not be well recognized, are also becoming possible due to recent advances in both experimental design and data collection technology.To make these new and unique applications of synchrotron soft X-rays available to the APS General User Community, a UGA-APS Pilot Program in this area was initiated in January 2016 at SER-CAT, Advanced Photon Source (APS), Argonne National Laboratory.Phase I of the Pilot Program was focused on fine-tuning the SER-CAT facility for Native-SAD that was reported at the 2016 ACA meeting in Denver.In this presentation, we are focusing on Phase II of the Pilot Program -the use of wavelength-dependent data for the chromatic exploration of uncharted biophysical/biochemical aspects of metals/ions in protein crystals.The theoretical basis and test results, as well as the procedures for accessing the Pilot Program through the General User portal, will be discussed.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

Nat. Methods 12, 131–133 (2015); published online 15 December 2014; corrected after print 6 February 2015 In the version of this article initially published, the Hendrickson formula in the Figure 2 legend incorrectly had (2NA/NP)1/2 divided by (f''/Zeff); these terms should have been multiplied. Theerror has been corrected in the HTML and PDF versions of the article.

The structures of several Bacillus thuringiensis (Bt) insecticidal crystal proteins have been determined by crystallographic methods and a close relationship has been explicated between specific toxicities and conserved three-dimensional architectures. In this study, as a representative of the coleopteran- and hemipteran-specific Cry51A group, the complete structure of Cry51Aa1 protoxin has been determined by X-ray crystallography at 1.65 Å resolution. This is the first report of a coleopteran-active Bt insecticidal toxin with high structural similarity to the aerolysin-type β-pore forming toxins (β-PFTs). Moreover, study of featured residues and structural elements reveal their possible roles in receptor binding and pore formation events. This study provides new insights into the action of aerolysin-type β-PFTs from a structural perspective, and could be useful for the control of coleopteran and hemipteran insect pests in agricultures.

Pirin is a nuclear nonheme Fe protein of unknown function present in all human tissues. Here we describe that pirin may act as a redox sensor for the nuclear factor κB (NF-κB) transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to proinflammatory signals and controls the expression of a vast array of genes involved in immune and stress responses. Pirin’s regulatory effect was tested with several metals and at different oxidations states, and our spectroscopic results show that only the ferric form of pirin substantially facilitates binding of NF-κB proteins to target κB genes, a finding that suggests that pirin performs a redox-sensing role in NF-κB regulation. The molecular mechanism of such a metal identity- and redox state-dependent regulation is revealed by our structural studies of pirin. The ferrous and ferric pirin proteins differ only by one electron, yet they have distinct conformations. The Fe center is shown to play an allosteric role on an R-shaped surface area that has two distinct conformations based on the identity and the formal redox state of the metal. We show that the R-shaped area composes the interface for pirin-NF-κB binding that is responsible for modulation of NF-κB’s DNA-binding properties. The nonheme Fe protein pirin is proposed to serve as a reversible functional switch that enables NF-κB to respond to changes in the redox levels of the cell nucleus.

Although the crystal structure of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase from Pseudomonas fluorescens was solved as a dimer, this enzyme is a mixture of monomer, dimer, and higher order structures in solution. In this work, we found that the dimeric state, not the monomeric state, is the functionally active form. Two conserved arginine residues are present in the active site: Arg-51 and an intruding Arg-239* from the neighboring subunit. In this study, they were each mutated to alanine and lysine, and all four mutants were catalytically inactive. The mutants were also incapable of accommodating pyridine-2,6-dicarboxylic acid, a competitive inhibitor of the native enzyme, suggesting that the two Arg residues are involved in substrate binding. It was also observed that the decarboxylase activity was partially recovered in a heterodimer hybridization experiment when inactive R51(A/K) and R239(A/K) mutants were mixed together. Of the 20 crystal structures obtained from mixing inactive R51A and R239A homodimers that diffracted to a resolution lower than 3.00 Å, two structures are clearly R51A/R239A heterodimers and belong to the C2 space group. They were refined to 1.80 and 2.00 Å resolutions, respectively. Four of the remaining crystals are apparently single mutants and belong to the P42212 space group. In the heterodimer structures, one active site is shown to contain dual mutation of Ala-51 and Ala-239*, whereas the other contains the native Arg-51 and Arg-239* residues, identical to the wild-type structure. Thus, these observations provide the foundation for a molecular mechanism by which the oligomerization state of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase could regulate the enzyme activity.

Dual-specificity phosphatases (DUSPs) play an important role in regulating cellular signalling pathways governing cell growth, differentiation and apoptosis. Human DUSP26 inhibits the apoptosis of cancer cells by dephosphorylating substrates such as p38 and p53. High-resolution crystal structures of the DUSP26 catalytic domain (DUSP26-C) and its C152S mutant [DUSP26-C (C152S)] have been determined at 1.67 and 2.20 Å resolution, respectively. The structure of DUSP26-C showed a novel type of domain-swapped dimer formed by extensive crossover of the C-terminal α7 helix. Taken together with the results of a phosphatase-activity assay, structural comparison with other DUSPs revealed that DUSP26-C adopts a catalytically inactive conformation of the protein tyrosine phosphate-binding loop which significantly deviates from that of canonical DUSP structures. In particular, a noticeable difference exists between DUSP26-C and the active forms of other DUSPs at the hinge region of a swapped C-terminal domain. Additionally, two significant gaps were identified between the catalytic core and its surrounding loops in DUSP26-C, which can be exploited as additional binding sites for allosteric enzyme regulation. The high-resolution structure of DUSP26-C may thus provide structural insights into the rational design of DUSP26-targeted anticancer drugs.

The previously reported crystal structures of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)(3)(Asp)(OH(2)) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescens (PfACMSD). Site-directed mutagenesis of His228 to tyrosine and glycine in this study results in a complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in the metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal charge transfer. Co(II)-substituted H228Y ACMSD is brown in color and exhibits an electron paramagnetic resonance spectrum showing a high-spin Co(II) center with a well-resolved (59)Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of as-isolated Fe-H228Y (2.8 Å) and Co-substituted (2.4 Å) and Zn-substituted H228Y (2.0 Å resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 Å) was also determined. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn-H228G) or disrupted (Zn-H228Y) in response to the His228 mutation. Together, these results highlight the importance of His228 for PfACMSD's metal specificity as well as maintaining a water molecule as a ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.

The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using a moderately diffracting crystal and 1.9 Å wavelength synchrotron X-rays. AF1382 was selected as a structural genomics target by the Southeast Collaboratory for Structural Genomics (SECSG) since sequence analyses showed that it did not belong to the Pfam-A database and thus could represent a novel fold. The structure was determined by exploiting longer wavelength X-rays and data redundancy to increase the anomalous signal in the data. AF1382 is a 95-residue protein containing five S atoms associated with four methionine residues and a single cysteine residue that yields a calculated Bijvoet ratio (ΔF(anom)/F) of 1.39% for 1.9 Å wavelength X-rays. Coupled with an average Bijvoet redundancy of 25 (two 360° data sets), this produced an excellent electron-density map that allowed 69 of the 95 residues to be automatically fitted. The S-SAD model was then manually completed and refined (R = 23.2%, R(free) = 26.8%) to 2.3 Å resolution (PDB entry 3o3k). High-resolution data were subsequently collected from a better diffracting crystal using 0.97 Å wavelength synchrotron X-rays and the S-SAD model was refined (R = 17.9%, R(free) = 21.4%) to 1.85 Å resolution (PDB entry 3ov8). AF1382 has a winged-helix-turn-helix structure common to many DNA-binding proteins and most closely resembles the N-terminal domain (residues 1-82) of the Rio2 kinase from A. fulgidus, which has been shown to bind DNA, and a number of MarR-family transcriptional regulators, suggesting a similar DNA-binding function for AF1382. The analysis also points out the advantage gained from carrying out data reduction and structure determination on-site while the crystal is still available for further data collection.

A multi-dataset (MDS) data-collection strategy is proposed and analyzed for macromolecular crystal diffraction data acquisition. The theoretical analysis indicated that the MDS strategy can reduce the standard deviation (background noise) of diffraction data compared with the commonly used single-dataset strategy for a fixed X-ray dose. In order to validate the hypothesis experimentally, a data-quality evaluation process, termed a readiness test of the X-ray data-collection system, was developed. The anomalous signals of sulfur atoms in zinc-free insulin crystals were used as the probe to differentiate the quality of data collected using different data-collection strategies. The data-collection results using home-laboratory-based rotating-anode X-ray and synchrotron X-ray systems indicate that the diffraction data collected with the MDS strategy contain more accurate anomalous signals from sulfur atoms than the data collected with a regular data-collection strategy. In addition, the MDS strategy offered more advantages with respect to radiation-damage-sensitive crystals and better usage of rotating-anode as well as synchrotron X-rays.

A high-throughput crystallization-to-structure pipeline for structural genomics has been developed at Advanced Protein Crystallography Research Group (APCR-group) of RIKEN SPring-8 Center, Japan.The structure determination pipeline includes three elemental technologies for automation developed in RIKEN SPring-8 Center: the automated crystallization and observation robot system TERA; X-ray diffraction data collection using the SPring-8 Precise Automatic Cryosample Exchanger SPACE; automatic X-ray data analysis from phasing to model checking/revision using the Package of Expert Researcher's Operation Network PERON.During five years from 2002, 234 of cryoloop-mountable crystals, 175 of diffraction data sets and 149 of refined structures have been produced from 437 of purified proteins in APCR-group, by seven researchers with assistance of the developed pipeline.Used protocols in this pipeline will be introduced.