Adenine recognition: a motif present in ATP-, CoA-, NAD-, NADP-, and FAD-dependent proteins.

Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Influence of rotational energy barriers to the conformational search of protein loops in molecular dynamics and ranking the conformations.

An adjustable-barrier dihedral angle potential was added as an extension to a novel, previously presented soft-core potential to study its contribution to the efficacy of the search of the conformational space in molecular dynamics. As opposed to the conventional soft-core potential functions, the leading principle in the design of the new soft-core potential, as well as of its extension, the soft-core and adjustable-barrier dihedral angle (SCADA) potential (referred as the SCADA potential), was to maintain the main equilibrium properties of the original force field. This qualifies the methods for a variety of a priori modeling problems without need for additional restraints typically required with the conventional soft-core potentials. In the present study, the different potential energy functions are applied to the problem of predicting loop conformations in proteins. Comparison of the performance of the soft-core and SCADA potential showed that the main hurdles for the efficient sampling of the conformational space of (loops in) proteins are related to the high-energy barriers caused by the Lennard-Jones and Coulombic energy terms, and not to the rotational barriers, although the conformational search can be further enhanced by lowering the rotational barriers of the dihedral angles. Finally, different evaluation methods were studied and a few promising criteria found to distinguish the near-native loop conformations from the wrong ones.     

Model of three-dimensional structure of vitamin D receptor and its binding mechanism with 1alpha,25-dihydroxyvitamin D(3).

Comparative modeling of the vitamin D receptor three-dimensional structure and computational docking of 1alpha,25-dihydroxyvitamin D(3) into the putative binding pocket of the two deletion mutant receptors: (207-423) and (120-422, Delta [164-207]) are reported and evaluated in the context of extensive mutagenic analysis and crystal structure of holo hVDR deletion protein published recently. The obtained molecular model agrees well with the experimentally determined structure. Six different conformers of 1alpha,25-dihydroxyvitamin D(3) were used to study flexible docking to the receptor. On the basis of values of conformational energy of various complexes and their consistency with functional activity, it appears that 1alpha,25-dihydroxyvitamin D(3) binds the receptor in its 6-s-trans form. The two lowest energy complexes obtained from docking the hormone into the deletion protein (207-423) differ in conformation of ring A and orientation of the ligand molecule in the VDR pocket. 1alpha,25-Dihydroxyvitamin D(3) possessing the A-ring conformation with axially oriented 1alpha-hydroxy group binds receptor with its 25-hydroxy substituent oriented toward the center of the receptor cavity, whereas ligand possessing equatorial conformation of 1alpha-hydroxy enters the pocket with A ring directed inward. The latter conformation and orientation of the ligand is consistent with the crystal structure of hVDR deletion mutant (118-425, Delta [165-215]). The lattice model of rVDR (120-422, Delta [164-207]) shows excellent agreement with the crystal structure of the hVDR mutant. The complex obtained from docking the hormone into the receptor has lower energy than complexes for which homology modeling was used. Thus, a simple model of vitamin D receptor with the first two helices deleted can be potentially useful for designing a general structure of ligand, whereas the advanced lattice model is suitable for examining binding sites in the pocket.     

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.     

Structural implications of drug-resistant mutants of HIV-1 protease: high-resolution crystal structures of the mutant protease/substrate analogue complexes

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-A resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wild-type are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D.     

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.     

Structure of a legume lectin from the bark of Robinia pseudoacacia and its complex with N-acetylgalactosamine

The structure of the bark lectin RPbAI (isoform A4) from Robinia pseudoacacia has been determined by protein crystallography both in the free form and complexed with N-acetylgalactosamine. The free form is refined at 1.80 A resolution to an R-factor of 18.9% whereas the complexed structure has an R-factor of 19.7% at 2.05 A resolution. Both structures are compared to each other and to other available legume lectin structures. The polypeptide chains of the two structures exhibit the characteristic legume lectin tertiary fold. The quaternary structure resembles that of the Phaseolus vulgaris lectin, the soybean agglutinin, and the Dolichos biflorus lectin, but displays some unique features leading to the extreme stability of this lectin.     

Elucidating protein secondary structures using alpha-carbon recurrence quantifications.

Secondary structures of proteins were studied by recurrence quantification analysis (RQA). High-resolution, 3-dimensional coordinates of alpha-carbon atoms comprising a set of 68 proteins were downloaded from the Protein Data Bank. By fine-tuning four recurrence parameters (radius, line, residue, separation), it was possible to establish excellent agreement between percent contribution of alpha-helix and beta-sheet structures determined independently by RQA and that of the DSSP algorithm (Define Secondary Structure of Proteins). These results indicate that there is an equivalency between these two techniques, which are based upon totally different pattern recognition strategies. RQA enhances qualitative contact maps by quantifying the arrangements of recurrent points of alpha carbons close in 3-dimensional space. For example, the radius was systematically increased, moving the analysis beyond local alpha-carbon neighborhoods in order to capture super-secondary and tertiary structures. However, differences between proteins could only be detected within distances up to about 6-11 A, but not higher. This result underscores the complexity of alpha-carbon spacing when super-secondary structures appear at larger distances. Finally, RQA-defined secondary structures were found to be robust against random displacement of alpha carbons upwards of 1 A. This finding has potential import for the dynamic functions of proteins in motion.     

Molecular dynamics studies on HIV-1 protease drug resistance and folding pathways

Drug resistance to HIV-1 protease involves the accumulation of multiple mutations in the protein. We investigate the role of these mutations by using molecular dynamics simulations that exploit the influence of the native-state topology in the folding process. Our calculations show that sites contributing to phenotypic resistance of FDA-approved drugs are among the most sensitive positions for the stability of partially folded states and should play a relevant role in the folding process. Furthermore, associations between amino acid sites mutating under drug treatment are shown to be statistically correlated. The striking correlation between clinical data and our calculations suggest a novel approach to the design of drugs tailored to bind regions crucial not only for protein function, but for folding as well.     

Structural basis of oncogenic activation caused by point mutations in the kinase domain of the MET proto-oncogene: modeling studies

Missense mutations in the tyrosine kinase domain of the MET proto-oncogene occur in selected cases of papillary renal carcinoma. In biochemical and biological assays, these mutations produced constitutive activation of the MET kinase and led to tumor formation in nude mice. Some mutations caused transformation of NIH 3T3 cells. To elucidate the mechanism of ligand-independent MET kinase activation by point mutations, we constructed several 3D models of the wild-type and mutated MET catalytic core domains. Analysis of these structures showed that some mutations (e.g., V1110I, Y1248H/D/C, M1268T) directly alter contacts between residues from the activation loop in its inhibitory conformation and those from the main body of the catalytic domain; others (e.g., M1149T, L1213V) increase flexibility at the critical points of the tertiary structure and facilitate subdomain movements. Mutation D1246N plays a role in stabilizing the active form of the enzyme. Mutation M1268T affects the S+1 and S+3 substrate-binding pockets. Models implicate that although these changes do not compromise the affinity toward the C-terminal autophosphorylation site of the MET protein, they allow for binding of the substrate for the c-Abl tyrosine kinase. We provide biochemical data supporting this observation. Mutation L1213V affects the conformation of Tyr1212 in the active form of MET. Several somatic mutations are clustered at the surface of the catalytic domain in close vicinity of the probable location of the MET C-terminal docking site for cytoplasmic effectors.     

Sm60, a mannose-binding protein from Schistosoma mansoni with inflammatory property

We demonstrate here that a mannose-binding protein from Schistosoma mansoni, termed Sm60, was recovered in the mannose-eluted fraction (Man(+)) upon affinity chromatography on immobilised mannose of the soluble antigen fraction from adult worm tegument and cercariae. Sm60 was detected in the Man(+) fraction as a prominent doublet with an apparent molecular mass of 60-66 kDa by SDS-PAGE and appeared as a single band with a pI of approximately 6.9 by isoelectrofocusing. Sm60 was also detected in preparations of schistosomula extract and soluble egg antigens using a mouse polyclonal anti-Sm60 serum on immunoblotting assay. This antiserum demonstrated that Sm60 was localised on the tegument of S. mansoni adult worm. In order to determine the role of Sm60 in host-parasite interactions, we showed that Sm60 induced in vitro migration of human neutrophil in a dose-dependent manner and in vitro mast cell degranulation. Sm60 triggered these activities through its carbohydrate-binding site, since these activities were selectively inhibited by 0.2 M D-mannose, but not by 0.2 M D-galactose. Furthermore, Sm60 induced in vivo neutrophil migration. In contrast, mast cell-depleted rats presented a significant reduction of the neutrophil migration induced by Sm60 as compared with non-depleted controls. These data suggest that in vivo neutrophil migration induced by Sm60 is modulated by mast cell-dependent mechanisms. Sm60 might play a key role in the host-parasite interaction, and its characterization opens perspective to examine the role of this molecule in the biology of S. mansoni.     

Tryptophanase in aqueous methanol: the solvent effects and a probable mechanism of the hydrophobic control of substrate specificity

To shed light on the mechanism of hydrophobic control in reactions of microbial tryptophanase the direct effect of the solvent hydrophobicity on affinities of amino acid inhibitors was first examined. Values of inhibition constants (K_i) for a variety of amino acids were determined in 37.5% aqueous methanol, and no general correlation between the change of K_i, on passing from water to aqueous methanol, and amino acid hydrophobicity was found. The solvent effects on the separate stages of the external aldimine formation (K_D) and deprotonation to form a quinonoid intermediate (K_q) were determined for the reactions of tryptophanase with 2-oxindolyl- -alanine and -alanine by stopped-flow technique. For 2-oxindolyl- -alanine, which is a close transition-state analogue for the enzyme reaction with natural substrate, the decrease in the affinity in aqueous methanol is associated exclusively with the α-proton abstraction stage but not with the preceding formation of external aldimine. We conclude that the environment of amino acid side chains in the active site cannot be considered to be permanently hydrophobic irrespective of the bound amino acid. We suggest that complexes of tryptophanase with amino acids may exist either in a hydrophobic, presumably “closed”, conformation, where bound amino acids are isolated from the solvent, or in an accesible to solvent, “open”, conformation, depending on the structure of the bound amino acid and stage of the catalytic mechanism. For 2-oxindolyl- -alanine the transfer from an open to a closed conformation probably accompanies deprotonation of the external aldimine. The change of the active site hydrophobicity may provide an efficient way of modulating the relative acid–base properties of the catalytic groups to ensure the movement of protons in the “correct” direction depending on the elementary stage of catalysis.     

我国影像临床试验注册情况及其对科研管理的启示

???? 目的 分析和探讨我国影像医学临床试验注册现状及存在的问题。方法 收集中国临床试验注册中心2008—2012年的临床试验注册数据,使用SPSS16.0软件进行统计分析,组间构成比比较采用Pearson χ²检验。结果 5年间我国影像医学临床试验注册数呈明显上升趋势。2011—2012年注册试验的影像手段构成与2008—2010年注册试验不同, P=0.037,MRI（从18.18%到32.73%）、CT（从9.09%到18.18%）试验所占比例明显升高。影像医学临床试验采用诊断试验设计的比例最高,占37.98%,明显高于平均水平3.45%,P<0.001。结论 我国影像临床试验的管理逐渐规范,临床试验的科学性和可信度随之提高。加强临床试验预注册、完善试验设计是提高影像医学试验管理水平的重要措施。     

Metabolic Engineering for Microbial Production of Aromatic Amino Acids and Derived Compounds

Metabolic engineering to design and construct microorganisms suitable for the production of aromatic amino acids and derivatives thereof requires control of a complicated network of metabolic reactions that partly act in parallel and frequently are in rapid equilibrium. Engineering the regulatory circuits, the uptake of carbon, the glycolytic pathway, the pentose phosphate pathway, and the common aromatic amino acid pathway as well as amino acid importers and exporters that have all been targeted to effect higher productivities of these compounds are discussed.     

临床科研管理系统在医院临床研究中的价值分析

目的 探讨临床科研管理系统在临床科室科研管理工作中的重要作用及推广价值。方法 使用SPSS15.0软件对数据进行统计分析,入组率比较采用χ²检验。结果 采用临床科研管理系统后,前3个月入组率不断升高,然后持续稳定在较高水平,前3个月的入组率（18.83%）明显低于后12个月的入组率（28.22%）,差异有统计学意义（P = 0.006）。目前已经有15项在研课题登记在案,科室科研氛围良好。结论 推行临床科研管理体系后,科研工作在数量和质量上均有了长足的发展和进步,在医院中具有推广价值。     

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.     

Production of functional recombinant tyrosine hydroxylase by the BPV-1 expression plasmids in the cell cultures

Bovine papilloma virus type-1 (BPV-1)-based expression plasmids TkBPVTH and CGalBPVTH encoding the rat tyrosine hydroxylase (TH) enzyme have been designed for the development of gene therapy for experimental Parkinson's disease. The aim of the present work was to examine the transfection of BPVTH plasmids to express a dopaminergic transgene in the monkey CV1-P fibroblast, rat C6 glioma and human NHA astrocyte cell cultures. The biological function of the transgene was estimated by analyzing the production of recombinant TH mRNA and protein, and the synthesis of L-dopa and dopamine. The highest transfection efficiency was obtained using TkBPVTH plasmids (5 microg). Furthermore, the expression of TkBPVTH plasmids was associated with significant synthesis of TH enzyme and L-dopa in the C6 and NHA cell cultures.     

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.