Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii.

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.

Crystal structure of a hypothetical protein, TTHA0829 from Thermus thermophilus HB8, composed of cystathionine-ß-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains.

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-ß-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four ß-strands and two α-helices in a ßαßßαß motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.

Atomic visualization of a flipped-back conformation of bisected glycans bound to specific lectins.

Glycans normally exist as a dynamic equilibrium of several conformations. A fundamental question concerns how such molecules bind lectins despite disadvantageous entropic loss upon binding. Bisected glycan, a glycan possessing bisecting N-acetylglucosamine (GlcNAc), is potentially a good model for investigating conformational dynamics and glycan-lectin interactions, owing to the unique ability of this sugar residue to alter conformer populations and thus modulate the biological activities. Here we analyzed bisected glycan in complex with two unrelated lectins, Calsepa and PHA-E. The crystal structures of the two complexes show a conspicuous flipped back glycan structure (designated 'back-fold' conformation), and solution NMR analysis also provides evidence of 'back-fold' glycan structure. Indeed, statistical conformational analysis of available bisected and non-bisected glycan structures suggests that bisecting GlcNAc restricts the conformations of branched structures. Restriction of glycan flexibility by certain sugar residues may be more common than previously thought and impinges on the mechanism of glycoform-dependent biological functions.

Crystal structures and ligand binding of PurM proteins from Thermus thermophilus and Geobacillus kaustophilus.

Crystal structures of 5-aminoimidazole ribonucleotide (AIR) synthetase, also known as PurM, from Thermus thermophilus (Tt) and Geobacillus kaustophilus (Gk) were determined. For TtPurM, the maximum resolution was 2.2 Å and the space group was P21212 with four dimers in an asymmetric unit. For GkPurM, the maximum resolution was 2.2 Å and the space group was P21212 with one monomer in asymmetric unit. The biological unit is dimer for both TtPurM and GkPurM and the dimer structures were similar to previously determined structures of PurM in general. For TtPurM, ∼50 residues at the amino terminal were disordered in the crystal structure whereas, for GkPurM, the corresponding region covered the ATP-binding site forming an α helix in part, suggesting that the N-terminal region of PurM changes its conformation upon binding of ligands. FGAM binding site was predicted by the docking simulation followed by the MD simulation based on the SO4 (2-) binding site found in the crystal structure of TtPurM.

Discovery, primary, and crystal structures and capacitation-related properties of a prostate-derived heparin-binding protein WGA16 from boar sperm.

Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface.

Structural basis for multiple sugar recognition of Jacalin-related human ZG16p lectin.

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related ß-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1-3Man and Glcß1-3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using (15)N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.

Structures and reaction mechanisms of the two related enzymes, PurN and PurU.

The crystal structures of glycinamide ribonucleotide transformylases (PurNs) from Aquifex aeolicus (Aa), Geobacillus kaustophilus (Gk) and Symbiobacterium toebii (St), and of formyltetrahydrofolate hydrolase (PurU) from Thermus thermophilus (Tt) were determined. The monomer structures of the determined PurN and PurU were very similar to the known structure of PurN, but oligomeric states were different; AaPurN and StPurN formed dimers, GkPurN formed monomer and PurU formed tetramer in the crystals. PurU had a regulatory ACT domain in its N-terminal side. So far several structures of PurUs have been determined, yet, the mechanisms of the catalysis and the regulation of PurU have not been elucidated. We, therefore, modelled ligand-bound structures of PurN and PurU, and performed molecular dynamics simulations to elucidate the reaction mechanisms. The evolutionary relationship of the two enzymes is discussed based on the comparisons of the structures and the catalytic mechanisms.

Structure of N-formylglycinamide ribonucleotide amidotransferase II (PurL) from Thermus thermophilus HB8.

The crystal structure of PurL from Thermus thermophilus HB8 (TtPurL; TTHA1519) was determined in complex with an adenine nucleotide, PO(4)(3-) and Mg(2+) at 2.35 Å resolution. TtPurL consists of 29 α-helices and 28 ß-strands, and one loop is disordered. TtPurL consists of four domains, A1, A2, B1 and B2, and the structures of the A1-B1 and A2-B2 domains were almost identical to each other. Although the sequence identity between TtPurL and PurL from Thermotoga maritima (TmPurL) is higher than that between TtPurL and the PurL domain of the large PurL from Salmonella typhimurium (StPurL), the secondary structure of TtPurL is much more similar to that of StPurL than to that of TmPurL.

Structural insights into recognition of triple-helical beta-glucans by an insect fungal receptor.

The innate ability to detect pathogens is achieved by pattern recognition receptors, which recognize non-self-components such as ß1,3-glucan. ß1,3-Glucans form a triple-helical structure stabilized by interchain hydrogen bonds. ß1,3-Glucan recognition protein (ßGRP)/gram-negative bacteria-binding protein 3 (GNBP3), one of the pattern recognition receptors, binds to long, structured ß1,3-glucan to initiate innate immune response. However, binding details and how specificity is achieved in such receptors remain important unresolved issues. We solved the crystal structures of the N-terminal ß1,3-glucan recognition domain of ßGRP/GNBP3 (ßGRP-N) in complex with the ß1,3-linked glucose hexamer, laminarihexaose. In the crystals, three structured laminarihexaoses simultaneously interact through six glucose residues (two from each chain) with one ßGRP-N. The spatial arrangement of the laminarihexaoses bound to ßGRP-N is almost identical to that of a ß1,3-glucan triple-helical structure. Therefore, our crystallographic structures together with site-directed mutagenesis data provide a structural basis for the unique recognition by such receptors of the triple-helical structure of ß1,3-glucan.

Crystal structures of human secretory proteins ZG16p and ZG16b reveal a Jacalin-related ß-prism fold.

ZG16p is a secretory protein that mediates condensation-sorting of pancreatic enzymes to the zymogen granule membrane in pancreatic acinar cells. ZG16p interacts with glycosaminoglycans and the binding is considered to be important for condensation-sorting of pancreatic enzymes. ZG16b/PAUF, a paralog of ZG16p, has recently been found to play a role in gene regulation and cancer metastasis. However, the detailed functions of ZG16p and ZG16b remain to be clarified. Here, in order to obtain insights into structure-function relationships, we conducted crystallographic studies of human ZG16p lectin as well as its paralog, ZG16b, and determined their crystal structures at 1.65 and 2.75 Å resolution, respectively. ZG16p has a Jacalin-related ß-prism fold, the first to be reported among mammalian lectins. The putative sugar-binding site of ZG16p is occupied by a glycerol molecule, mimicking the mannose bound to Jacalin-related mannose-binding-type plant lectins such as Banlec. ZG16b also has a ß-prism fold, but some amino acid residues of the putative sugar-binding site differ from those of the mannose-type binding site suggesting altered preference. A positively charged patch, which may bind sulfated groups of the glycosaminoglycans, is located around the putative sugar-binding site of ZG16p and ZG16b. Taken together, we suggest that the sugar-binding site and the adjacent basic patch of ZG16p and ZG16b cooperatively form a functional glycosaminoglycan-binding site.

Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of IMP or GMP from alpha-D-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. Structures of HGPRTase from Thermus thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP have been determined at 2.1, 1.9 and 2.2 A resolution, respectively. The overall fold of the IMP complex was similar to that of the unliganded form, but the main-chain and side-chain atoms of the active site moved to accommodate IMP. The overall folds of the IMP and GMP complexes were almost identical to each other. Structural comparison of the T. thermophilus HB8 enzyme with 6-oxopurine PRTases for which structures have been determined showed that these enzymes can be tentatively divided into groups I and II and that the T. thermophilus HB8 enzyme belongs to group I. The group II enzymes are characterized by an N-terminal extension with additional secondary elements and a long loop connecting the second alpha-helix and beta-strand compared with the group I enzymes.

Crystal structures of glycinamide ribonucleotide synthetase, PurD, from thermophilic eubacteria.

Glycinamide ribonucleotide synthetase (GAR-syn, PurD) catalyses the second reaction of the purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine and ATP to glycinamide ribonucleotide (GAR), ADP and Pi. In the present study, crystal structures of GAR-syn's from Thermus thermophilus, Geobacillus kaustophilus and Aquifex aeolicus were determined in apo forms. Crystal structures in ligand-bound forms were also determined for G. kaustophilus and A. aeolicus proteins. In general, overall structures of GAR-syn's are similar to each other. However, the orientations of the B domains are varied among GAR-syn's and the MD simulation suggested the mobility of the B domain. Furthermore, it was demonstrated that the B loop in the B domain fixes the position of the ß- and γ- phosphate groups of the bound ATP. The structures of GAR-syn's and the bound ligands were compared with each other in detail, and structures of GAR-syn's with full ligands, as well as the possible reaction mechanism, were proposed.

Crystallization screening test for the whole-cell project on Thermus thermophilus HB8.

It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.

Crystal structure of the YdjC-family protein TTHB029 from Thermus thermophilus HB8: structural relationship with peptidoglycan N-acetylglucosamine deacetylase.

The YdjC-family protein is widely distributed, from human to bacteria, but so far no three-dimensional structure and functional analysis of this family of proteins has been reported. We determined the three-dimensional structure of the YdjC homolog TTHB029 at a resolution of 2.9A. The overall structure of the monomer consists of (betaalpha)-barrel fold forming a homodimer. Asp21, His60, and His127 residues coordinate to Mg(2+) as a possible active site. TTHB029 shows structural similarity to the peptidoglycan N-acetylglucosamine deacetylase from Streptococcus pneumoniae (SpPgdA). The active site groove of SpPgdA includes the Zn(2+) coordinated to Asp276, His326, and His330. Despite the low sequence identity, metal-binding residues of Asp-His-His were conserved among the two enzymes. There were definitive differences, however, in that one of the histidines of the metal-binding site was substituted for the other histidine located on the other loop. Moreover, these important metal-binding residues and the residues of the presumed active site are fully conserved in YdjC-family protein.