Crystal structure of UDP-galactose 4-epimerase from the hyperthermophilic archaeon Pyrobaculum calidifontis

The crystal structure of a highly thermostable UDP-galactose 4-epimerase (GalE) from the hyperthermophilic archaeon Pyrobaculum calidifontis was determined at a resolution of 1.8 Å. The asymmetric unit contained one subunit, and the functional dimer was generated by a crystallographic two-fold axis. Each monomer consisted of a Rossmann-fold domain with NAD bound and a carboxyl terminal domain. The overall structure of P. calidifontis GalE showed significant similarity to the structures of the GalEs from Escherichia coli, human and Trypanosoma brucei. However, the sizes of several surface loops were markedly smaller in P. calidifontis GalE than the corresponding loops in the other enzymes. Structural comparison revealed that the presence of an extensive hydrophobic interaction at the subunit interface is likely the main factor contributing to the hyperthermostability of the P. calidifontis enzyme. Within the NAD-binding site of P. calidifontis GalE, a loop (NAD-binding loop) tightly holds the adenine ribose moiety of NAD. Moreover, a deletion mutant lacking this loop bound NAD in a loose, reversible manner. Thus the presence of the NAD-binding loop in GalE is largely responsible for preventing the release of the cofactor from the holoenzyme.     

Crystal structure of the mismatch-specific thymine glycosylase domain of human methyl-CpG-binding protein MBD4

Methyl-CpG (mCpG) binding domain protein 4 (MBD4) is a member of mammalian DNA glycosylase superfamily. It contains an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain, which is an important molecule believed to be involved in maintaining of genome stability. Herein, we determined the crystal structure of C-terminal glycosylase domain of human MBD4. And the structural alignments of other helix-hairpin-helix (HhH) DNA glycosylases show that the human MBD4 glycosylase domain has the similar active site and the catalytic mechanisms as others. But the different residues in the N-terminal of domain result in the change of charge distribution on the surface of the protein, which suggest the different roles that may relate some diseases.     

Crystal structure of chondroitin polymerase from Escherichia coli K4

Elongation of glycosaminoglycan chains, such as heparan and chondroitin, is catalyzed by bi-functional glycosyltransferases, for which both 3-dimensional structures and reaction mechanisms remain unknown. The bacterial chondroitin polymerase K4CP catalyzes elongation of the chondroitin chain by alternatively transferring the GlcUA and GalNAc moiety from UDP-GlcUA and UDP-GalNAc to the non-reducing ends of the chondroitin chain. Here, we have determined the crystal structure of K4CP in the presence of UDP and UDP-GalNAc as well as with UDP and UDP-GlcUA. The structures consisted of two GT-A fold domains in which the two active sites were 60 Å apart. UDP-GalNAc and UDP-GlcUA were found at the active sites of the N-terminal and C-terminal domains, respectively. The present K4CPstructures have provided the structural basis for further investigating the molecular mechanism of biosynthesis of chondroitin chain.     

Crystal and molecular structure of methyl 4-O-methyl-beta-D-ribo-hex-3-ulopyranoside

Methyl 4-O-methyl-beta-D-ribo-hex-3-ulopyranoside (2), a model compound for partially oxidized anhydroglucose units in cellulose, was crystallized from CHCl(3)/n-hexane by vapor diffusion to give colorless plates. Crystal structure determination revealed the monoclinic space group P2(1) with Z = 2C(8)H(14)O(6) and unit cell parameters of a = 8.404(2), b = 4.5716(10), c = 13.916(3)A, and beta = 107.467(4) degrees. The structure was solved by direct methods and refined to R = 0.0476 for 1655 reflections and 135 parameters. The hexulopyranoside occurs in a distorted chair conformation. Both hydroxyls are involved in hydrogen bonding and form zigzag bond chains along the b-axis. One of the two hydrogen bonds is bifurcated. The solid-state (13)C NMR spectrum of exhibits eight carbon resonances, with well-separated signals for the two methoxyls (1-OMe: 55.72 ppm, 4-OMe: 61.25 ppm) and a keto resonance with relatively large downfield shift (206.90 ppm). Differences in the C-4 and the methoxyls' chemical shifts in the solid and liquid states were found.     

Crystal structure of TbAlba1 from Trypanosoma brucei

Alba (Acetylation lowers binding affinity) domain is a small, dimeric nucleic acid-binding domain, which is widely distributed in archaea and numbers of eukaryotes. Alba domain containing proteins have been reported to be involved in many cellular processes, such as regulation of translation, maintaining genome stability, regulation of RNA processing and so on. In Trypanosoma brucei (T. brucei), there are four Alba proteins identified, which are named TbAlba1 to TbAlba4. However, the structure and function of TbAlba proteins are still unknown. Here, we solved the crystal structure of TbAlba1 to a resolution of 2.46 Å. TbAlba1 adopts a similar Alba-fold, which comprises of four β-strands (β1-β4) and three long α-helices (α1-α3). Furthermore, TbAlba1 displays some structural features quite different from other Alba proteins. These differences may imply the diverse biological roles of Alba family members.     

Crystal structure of the phospholipase A and acyltransferase 4 (PLAAT4) catalytic domain

Phospholipase A and Acyltransferase 4 (PLAAT4) is a class II tumor suppressor, that also plays a role as a restrictor of intracellular Toxoplasma gondii infection through restriction of parasitic vacuole size. The catalytic N-terminal domain (NTD) interacts with the C-terminal domain (CTD), which is important for sub-cellular targeting and enzymatic function. The dynamics of the NTD main (L1) loop and the L2(B6) loop adjacent to the active site, have been shown to be important regulators of enzymatic activity. Here, we present the crystal structure of PLAAT4 NTD, determined from severely intergrown crystals using automated, laser-based crystal harvesting and data reduction technologies. The structure showed the L1 loop in two distinct conformations, highlighting a complex network of interactions likely influencing its conformational flexibility. Ensemble refinement of the crystal structure recapitulates the major correlated motions observed in solution by NMR. Our analysis offers useful insights on millisecond dynamics based on the crystal structure, complementing NMR studies which preclude structural information at this time scale.     

Crystal structure and magnetic behaviour of a five-coordinate iron(III) complex of pyridoxal-4-methylthiosemicarbazone

The crystal structure and magnetic properties of a penta-coordinate iron(III) complex of pyridoxal-4-methylthiosemicarbazone, [Fe(H₂mthpy)Cl₂](CH₃C₆H₄SO₃), are reported. The synthesised ligand and the metal complex were characterised by spectroscopic methods (¹H NMR, IR, and mass spectroscopy), elemental analysis, and single crystal X-ray diffraction. The complex crystallises as dark brown microcrystals. The crystal data determined at 100(1) K revealed a triclinic system, space group (Z = 2). The ONSCl₂ geometry around the iron(III) atom is intermediate between trigonal bipyramidal and square pyramidal (τ = 0.40). The temperature dependence of the magnetic susceptibility (5-300 K) is consistent with a high spin Fe(III) ion (S = 5/2) exhibiting zero-field splitting. Interpretation of these data yielded: D = 0.34(1) cm⁻¹ and g = 2.078(3).     

Crystal and molecular structure of methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside

The cellulose model compound methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside (6) was synthesised in high overall yield from methyl beta-D-cellobioside. The compound was crystallised from methanol to give colourless prisms, and the crystal structure was determined. The monoclinic space group is P2(1) with Z=2 and unit cell parameters a=6.6060 (13), b=14.074 (3), c=9.3180 (19) A, beta=108.95(3) degrees. The structure was solved by direct methods and refined to R=0.0286 for 2528 reflections. Both glucopyranoses occur in the 4C(1) chair conformation with endocyclic bond angles in the range of standard values. The relative orientation of both units described by the interglycosidic torsional angles [phi (O-5' [bond] C-1' [bond] O-4 [bond] C-4) -89.1 degrees, Phi (C-1' [bond] O-4 [bond] C-4 [bond] C-5) -152.0 degrees] is responsible for the very flat shape of the molecule and is similar to those found in other cellodextrins. Different rotamers at the exocyclic hydroxymethyl group for both units are present. The hydroxymethyl group of the terminal glucose moiety displays a gauche-trans orientation, whereas the side chain of the reducing unit occurs in a gauche-gauche conformation. The solid state (13)C NMR spectrum of compound 6 exhibits all 14 carbon resonances. By using different cross polarisation times, the resonances of the two methyl groups and C-6 carbons can easily be distinguished. Distinct differences of the C-1 and C-4 chemical shifts in the solid and liquid states are found.     

Crystal structure of a family 4 uracil-DNA glycosylase from Thermus thermophilus HB8

Uracil-DNA glycosylase (UDG; EC 3.2.2.-) removes uracil from DNA to initiate DNA base excision repair. Since hydrolytic deamination of cytosine to uracil is one of the most frequent DNA-damaging events in all cells, UDG is an essential enzyme for maintaining the integrity of genomic information. For the first time, we report the crystal structure of a family 4 UDG from Thermus thermophilus HB8 (TthUDG) complexed with uracil, solved at 1.5 angstroms resolution. As opposed to UDG enzymes in its other families, TthUDG possesses a [4Fe-4S] cluster. This iron-sulfur cluster, which is distant from the active site, interacts with loop structures and has been suggested to be unessential to the activity but necessary for stabilizing the loop structures. In addition to the iron-sulfur cluster, salt-bridges and ion pairs on the molecular surface and the presence of proline on loops and turns is thought to contribute to the enzyme's thermostability. Despite very low levels of sequence identity with Escherichia coli and human UDGs (family 1) and E.coli G:T/U mismatch-specific DNA glycosylase (MUG) (family 2), the topology and order of secondary structures of TthUDG are similar to those of these distant relatives. Furthermore, the coordinates of the core structure formed by beta-strands are almost the same. Positive charge is distributed over the active-site groove, where TthUDG would bind DNA strands, as do UDG enzymes in other families. TthUDG recognizes uracil specifically in the same manner as does human UDG (family 1), rather than guanine in the complementary strand DNA, as does E.coli MUG (family 2). These results suggest that the mechanism by which family 4 UDGs remove uracils from DNA is similar to that of family 1 enzymes.     

Crystal structure of non-redox regulated SSADH from Escherichia coli

SSADH is involved in the final step of GABA degradation, converting SSA to succinic acid in the human mitochondrial matrix, and its activity is known to be regulated via ‘redox-switch modulation’ of the catalytic loop. We present the crystal structure of EcSSADH, revealing that the catalytic loop of EcSSADH, unlike that of human SSADH, does not undergo disulfide bond-mediated structural changes upon changes of environmental redox status. Subsequent redox change experiments using recombinant proteins confirm the non-redox regulation of this protein. Detailed structural analysis shows that a difference in the conformation of the connecting loop (β15-β16) causes the formation of a water molecule-mediated hydrogen bond network between the connecting loop and the catalytic loop in EcSSADH, making the catalytic loop of EcSSADH more rigid compared to that of human SSADH. The cytosolic localization of EcSSADH and the cellular function of the GABA shunt in E. coli might result in the non-redox mediated regulatory mechanisms of the protein.     

Crystal structure of prephenate dehydrogenase from Streptococcus mutans

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD⁺ shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between β6 and β7, which may be a reason for the high K_m values of PDH from Streptococcus mutans.     

Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus

We have determined a 2.6 Å resolution crystal structure of Pfu DNA polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus. Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The interaction of these two domains is known to coordinate the proofreading and polymerization activity of DNA polymerases, especially via H147 that is present within the loop (residues 144–158) of the exonuclease domain. In our structure of Pfu, however, E148 rather than H147 is located at better position to interact with the thumb domain. In addition, the structural analysis of Pfu and KOD1 shows that both the Y-GG/A and β-hairpin motifs of Pfu are found to differ with that of KOD1, and may explain differences in processivity. This information enables us to better understand the mechanisms of polymerization and proofreading of DNA polymerases.     

Crystal structure of scaffolding protein CheW from thermoanaerobacter tengcongensis

The crystal structure of the scaffolding protein CheW from Thermoanaerobacter tengcongensis (TtCheW) is reported with a resolution at 2.2A using molecular replacement. Based on the crystal structure TmCheA P4-P5-TmCheW from Thermotoga maritime reported by others, we modeled the TmCheA P4-P5-TtCheW complex and predicted that TtCheW is involved in a hydrophobic interaction with CheA, similar to that for TmCheW. We also found that the conserved motif "NxxGxIxP" from CheW plays an important role in CheA binding. The coincidence of the reported mutation sites related to CheW-MCP binding, and the predicted protein interaction region within the TtCheW molecule, suggest that CheW-MCP binding sites lie in the groove-shaped area between TtCheW and the CheA P4 domain within the assembled model.     

Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa

PilF is a requisite protein involved in the type 4 pilus biogenesis system from the Gram-negative human pathogenic bacteria, Pseudomonas aeruginosa. We determined the PilF structure at a 2.2A resolution; this includes six tandem tetratrico peptide repeat (TPR) units forming right-handed superhelix. PilF structure was similar to the heat shock protein organizing protein, which interacts with the C-terminal peptide of Hsp90 and Hsp70 via a concave Asn ladder in the inner groove of TPR superhelix. After simulated screening, the C-terminal pentapeptides of PilG, PilU, PilY, and PilZ proved to be a likely candidate binding to PilF, which are ones of 25 necessary components involved in the type 4 pilus biogenesis system. We proposed that PilF would be critical as a bridgehead in protein-protein interaction and thereby, PilF may bind a necessary molecule in type 4 pilus biogenesis system such as PilG, PilU, PilY, and PilZ.     

沙冬青AmLEA5基因的生物信息学分析及非生物胁迫下的表达模式

本文对用固相扣除杂交方法从低温驯化沙冬青克隆得到的AmLEA5基因进行分子特性和表达模式分析,生物信息学分析表明该基因编码一种第5族胚胎晚期发生丰富蛋白（LEA）。AmLEA5基因全长693 bp,含有1个297 bp的开放阅读框,编码98个氨基酸,预测AmLEA5的分子量为10.6 kDa,是一种亲水性蛋白,有多个磷酸化位点。密码子偏好性分析表明该基因略偏好于用A或T结尾的密码子。系统发生分析表明,AmLEA5蛋白与蒺藜苜蓿LEA（ACJ84182.1）亲缘关系最近。qRT-PCR结果显示AmLEA5的表达量在低温、干旱、盐和热胁迫条件下均有上调,尤其在低温胁迫后期富集量最高。亚细胞定位表明,用YFP标记的AmLEA5位于细胞质和细胞核内。一系列实验结果说明AmLEA5基因在沙冬青抵御非生物胁迫,尤其是在抵御低温胁迫机制中发挥重要作用。     

Crystal structure of SecB from Escherichia coli

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 A resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.     

Crystal structure of phospholipase PA2-Vb,a protease-activated receptor agonist from the Trimeresurus stejnegeri snake venom

Phospholipase A₂ (PLA₂) is an important component in snake venoms. Here, an acidic PLA₂, designated PA2-Vb was isolated from the Trimeresurus stejnegeri snake venom. PA2-Vb acts on a protease-activated receptor (PAR-1) to evoke Ca²⁺ release through the inositol 1,4,5-trisphosphate receptor (IP₃R) and induces mouse aorta contraction. PAR-1, phospholipase C and IP₃R inhibitors suppressed PA2-Vb-induced aorta contraction. The crystal structure reveals that PA2-Vb has the typical fold of most snake venom PLA₂. Several PEG molecules bond to a positively charged pocket. The finding offers a novel pharmacological basis of the structure for investigating the PAR-1 receptor and suggests potential applications for PA2-Vb in the vascular system.     

Crystal structure of a cyclomaltoheptaose-4-hydroxybiphenyl inclusion complex

The crystal structure of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with 4-hydroxybiphenyl was determined by single-crystal X-ray diffraction at 150K. The complex contains two cyclomaltoheptaose molecules, two 4-hydroxybiphenyl molecules, one ethanol molecule and fifteen water molecules in the asymmetric unit, and could be formulated as [2(C(42)H(70)O(35)).2(C(12)H(10)O).(C(2)H(6)O).15(H(2)O)]. It crystallized in the triclinic space group P1 with unit cell constants a=15.257(3), b=15.564(3), c=15.592(2)A, alpha=104.485(15) degrees , beta=101.066(14) degrees , gamma=104.330(17) degrees , V=3,343.6(10)A(3). In the crystal lattice, two beta-cyclodextrins form a head-to-head dimer jointed through hydrogen bonds. Two 4-hydroxybiphenyls were included in the dimer cavity with their hydroxyl groups protruding from two primary hydroxyl sides of the cyclodextrin molecules. The guest 4-hydroxybiphenyl molecules linked into a chain via a combination of an O-Hcdots, three dots, centeredO hydrogen bond and face-to-face pi-pi stacking of the phenyl rings. The crystal structure supports the calculation results indicating that the 2:2 inclusion complex formed by beta-cyclodextrin and 4-hydroxybiphenyl is the energetically favored structure.     

Crystal structure and microstructure of cholesteryl oleyl carbonate

The crystal structure as well as the microstructure, i.e., size and strain, of crystallites of cholesteryl oleyl carbonate was determined from X-ray powder diffraction data. The X-ray line broadening was analyzed through the refinement of TCH-pseudo-Voigt function parameters (isotropic effects) and the refinement of multipolar functions, i.e., symmetrized cubic harmonics (anisotropic effects). The crystal structure turns out to be primitive monoclinic, space group Pc, type I monolayer having two molecules per unit cell with parameters: a = 18.921 ± 0.006 Å, b = 12.952 ± 0.003 Å, c = 9.276 ± 0.002 Å and β = 91.32 ± 0.03°. The average size of a well ground specimen of crystallites was 60 nm. The average micro-strain, e.g., 45 × 10⁻⁴ has been tentatively attributed to fatty chain conformational disorder. The unit cell parameters, including the lamellar thickness, of COC crystal is very closely similar to those of another, structurally similar cholesterol ester, e.g., cholesteryl oleate (CO) crystal, space group P2₁, type II monolayer. Type I monolayer structure has been established for COC on the basis of the intensity calculations of the XRD profiles of both CO and COC. The dipolar and structural disorder in a 4:1 molar, binary mixture of CO and COC can be accommodated in an induced smectic phase with a lamellar thickness, which is nearly equal to that of pure CO or pure COC.