Structure of Bacteriophage T4 Endonuclease II Mutant E118A, a Tetrameric GIY-YIG Enzyme

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16 kDa, 136 aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P2₁ with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.     

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C^pro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C^pro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C^pro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3C^pro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4′ region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C^pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C^pro.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.     

Structure of the Complex between HER2 and an Antibody Paratope Formed by Side Chains from Tryptophan and Serine

Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 Å resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.     

Caffeic acid protects human peripheral blood lymphocytes against gamma radiation-induced cellular damage

In the present study, we investigated in vitro radioprotective potential of caffeic acid (CA), a naturally occurring catecholic acid against gamma radiation-induced cellular changes. Different concentrations of CA (5.5, 11, 22, 44, 66, and 88 microM) were incubated with lymphocytes for 30 min prior to gamma-irradiation, and micronuclei (MN) scoring and comet assay were performed to fix the effective concentration of CA against gamma-irradiation. Among all concentrations, 66 microM of CA showed the optimum protection by effectively decreasing the MN frequencies and comet attributes. From the above-mentioned results, 66 microM of CA was selected as the effective concentration and was further used to investigate its radioprotective efficacy. For that purpose, a separate experiment was carried out on the lymphocytes in which lymphocytes were preincubated with CA (66 microM) and were exposed to different doses of radiation (1, 2, 3, and 4 Gy). Genetic damage (MN, dicentric aberration, and comet attributes) and biochemical changes were measured. Gamma-irradiated lymphocytes showed a dose-dependent increase in the genetic damage and thiobarbituric acid reactive substances, accompanied by the significant decrease in the antioxidant status, whereas CA pretreatment positively modulated all the radiation-induced changes through its antioxidant potential. The current study demonstrates that CA is effective in protecting lymphocytes against radiation-induced toxicity and encourages further in vivo study to evaluate radioprotective efficacy of CA.     

人工同龄纯林冠层辐射场模拟模型II 应用与验证

 应用建立的人工同龄林辐射场模型对杉木(Cunninghamia lanceolata)人工林冠层辐射场进行了模拟,以实测的冠层辐射数据对模型进行了验证,对模型的不确定性以及叶面积密度和叶片散射的影响进行了计算和分析。结果表明,模拟和实测辐射场及日总量无显著差异,所建模型适于对杉木人工林冠层辐射的模拟。模型可能的误差来源包括树冠形状的不规则性、树冠大小和高低的不一致性、叶片在小范围内的簇生性等。考虑二维叶面积密度（LAD）分布的模拟结果优于平均LAD。叶片的散射辐射约占总辐射的3％～11％,且越靠近树冠里层     

Radical oxidation of the adenine moiety of nucleoside and DNA: 2-hydroxy-2'-deoxyadenosine is a minor decomposition product

A method involving high performance liquid chromatography (HPLC) separation associated with tandem mass spectrometry (MS/MS) detection in the multiple reaction monitoring mode was set-up for the measurement of 2-hydroxy-2'-deoxyadenosine (2-OHdAdo). This modified nucleoside, arising from the radical oxidation of 2'-deoxyadenosine (dAdo), has been described in the literature as a potential biological marker of the Fenton reaction. Using the specific and sensitive HPLC-MS/MS assay, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 4,6-diamino-5-formamidopyrimidine and 2-hydroxy-2'-deoxyadenosine (2-OHdAdo) were measured within 2'-deoxyadenosine and DNA solutions either exposed to γ-rays or treated under Fenton reaction conditions. It was found that the yield of 2-OHdAdo was low compared to that of 8-oxodAdo under most of the oxidative conditions studied. In particular and in contrast to previous works, the formation of 2-OHdAdo was shown to be a minor process both upon gamma irradiation and under Fenton reaction conditions. However, a significant yield of formation of 2-OHdAdo was observed either upon incubation with high concentrations of Fe 2+ ions in the absence of hydrogen peroxide or upon γ-radiolysis of a nucleoside solution in the presence of the copper/ ( o )-phenanthroline complex.     

Selective induction of mitogen-activated protein kinases in human lens epithelial cells by ultraviolet radiation

The present study investigates the effects of ultraviolet radiation (UVR) on mitogen-activated protein kinase (MAPK) activity in human lens epithelial (HLE) cells. Irradiation of HLE cells with ultraviolet B and ultraviolet C radiation activates the stress-response MAPK proteins, p38 and c-Jun NH(2)-terminal kinase (JNK), in a dose- and time-dependent manner, while the extracellular-regulated signal kinase (ERK 44/42) cascade was not altered by UVR exposure. Ultraviolet A radiation failed to elicit a MAPK response. UVR-induced MAPK activation does not require protein kinase C or phosphatidylinositol 3-kinase activity, suggesting that this is not a receptor-mediated event. Inhibition of ribosomal translation completely abolished UVR-induced MAPK activation, while treatment with the antioxidant, N-acetyl cysteine, and mild heat shock had no effect on this activation. These data demonstrate for the first time the selective activation of MAPK cascades in a lens epithelial cell line.