The Structure of Neplanocin C

Abstract

The molecular and crystal structure of neplanocin C(3), C₁₁H₁₃N₅O₄ M.W. = 279.26, has been determined by X-ray anlys?s. The space group is P2₁ with a=16.381(2), b=8.210(1), c=9.127(1) Å, β=105.31(1)° and z=4. The structure was solved by direct method, and least-squares refinement using 2093 reflections with |Fo|>3σ(F) led to the final R value of 0.0772. The sugar puckering of the two crystal-lographically independent molecules is C(2′)-exo-C(3′)-endo, and the torsion angles about the N(9)-C(1′) bond are 22.8(6) and 28.7(6)°, respectively (anti conformation).     

Structure and activity of C1r and C1s

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.     

Crystal Structure of Human Carbonic Anhydrase C

The three dimensional structure of human carbonic anhydrase C has been determined at 2.0 Å resolution. The active site has been identified by the binding of inhibitors and the location of the zinc ion.     

Structure and function of clostridial phospholipases C

A range of clostridial species produce phospholipases C. The zinc metallo phospholipases C have related sequences but different properties. All of these enzymes may be arranged, like alpha-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.     

Structure of C protein purified from cardiac muscle

C protein is a component of the thick filament of striated muscles. Although the function of C protein remains unknown, a variety of evidence suggests that C protein may regulate actin-myosin interaction or be involved in structural support or elasticity of the sarcomere. We have previously proposed (Hartzell, H. C., 1984, J. Gen. Physiol., 83:563-588) that C protein is involved in regulating twitch relaxation in cardiac muscle. To gain further insight into the function of C protein, we have studied the structure of C protein purified from chicken heart. C protein was purified from extracts of detergent-washed myofibrils by sequential hydroxylapatite and DEAE-Sephacel chromatography. C protein was judged greater than 95% pure by SDS PAGE. The polypeptide subunit had a molecular weight of 155,000 and the native molecule sedimented on linear sucrose or glycerol gradients at 4-5S. For electron microscopy, purified C protein was dialyzed and diluted into a volatile buffer in 50% glycerol, aspirated onto mica, dried under vacuum, and rotary platinum-shadowed. Replicas revealed particles of relatively homogeneous overall dimensions. Over half of the particles were V-shaped. The "arm" lengths of the V-shaped particles were 22 +/- 4.5 nm (SD). Gel filtration on Sephacryl S-300 demonstrated that purified C protein had a Stokes' radius of 5.07 nm. Measurements of viscosity gave an intrinsic viscosity of 16.5 cm3/g. These data are consistent with the electron microscopic data and suggest that C protein in heart muscle is asymmetric. The C protein molecule is large enough to extend from the surface of a thick filament to adjacent thin or thick filaments.     

Structure and organization of the C4 genes

This 200 000 Mr serum protein is coded for by at least two separate loci, C4A and C4B, which map in the HLA Class III region on chromosome 6 in man. Both loci are highly polymorphic with more than 30 alleles, including null alleles assigned to the two loci. The complete nucleotide sequence of a full length C4A cDNA clone and a substantial part of a C4b cDNA clone has shown class differences which can be used to synthesize nucleotide probes specific for C4A and C4B. Three C4 loci of approximately 16 kilobases each spaced by 10 kilobases have been identified in DNA from one individual and aligned 30 kilobases from the factor B gene by overlapping cloned genomic fragments from a cosmid library. Characterization of these genes by restriction mapping, nucleotide sequence analysis and hybridization with C4A and C4B specific synthetic oligonucleotides show that these genes are very similar.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

丙型肝炎病毒基因组结构及功能

丙型肝炎病毒（hepatitis C virus, HCV）是单股正链的RNA 病毒,全长为9.6 kb,包括1个大的开放阅读框（ORF）和两侧的5′,3′非编码区（UTRs）.核糖体通过进入HCV 5′UTR 端的内部核糖体进入位点（IRES）,将HCV基因组翻译成1个聚蛋白前体.前体聚蛋白被宿主和病毒的蛋白酶共同切割成为若干个具有独立功能的HCV蛋白,根据功能的不同分别命名为C、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A 和NS5B,它们不但在HCV的生活史中发挥着重要的作用,也影响着宿主细胞的信号传导、凋亡及物质代谢等一系列生化过程.近年来,随着HCV体外细胞摸型的不断发展,其病毒分子生物学方面的研究取得了很大的进展.本文从基因组结构及其编码的蛋白功能等方面阐述了HCV病毒的研究进展,为致病机理的研究及抗HCV药物的开发和疫苗研制等提供理论基础.     

Structure analysis of rimuene by 13C nmr spectroscopy

A diterpenoid forming the major component of the hydrocarbon fraction of the epicuticular wax of Richea continentis from Baw Baw Alpine Reserve is shown to be rimuene.     

Structure and Inhibition of Mouse Leukotriene C4 Synthase

Leukotriene (LT) C₄ synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA₄ and GSH to form the pro-inflammatory LTC₄, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhibitor and compared its efficiency and binding mode against the purified mouse and human isoenzymes, along with the enzymes’ steady-state kinetics. Although structural differences near the active site and along the C-terminal α-helix V suggest that the mouse and human LTC4S may function differently in vivo, our data indicate that mouse LTC4S will be a useful tool in future pharmacological research and drug development.     

Structure of the Janus-faced C2B domain of rabphilin

C2 domains are widespread protein modules that often occur as tandem repeats in many membrane-trafficking proteins such as synaptotagmin and rabphilin. The first and second C2 domains (C2A and C2B, respectively) have a high degree of homology but also specific differences. The structure of the C2A domain of synaptotagmin I has been extensively studied but little is known about the C2B domains. We have used NMR spectroscopy to determine the solution structure of the C2B domain of rabphilin. The overall structure of the C2B domain is very similar to that of other C2 domains, with a rigid beta-sandwich core and loops at the top (where Ca2+ binds) and the bottom. Surprisingly, a relatively long alpha-helix is inserted at the bottom of the domain and is conserved in all C2B domains. Our results, together with the Ca(2+)-independent interactions observed for C2B domains, indicate that these domains have a Janus-faced nature, with a Ca(2+)-binding top surface and a Ca(2+)-independent bottom surface.     

Structure and expression of the human cystatin C gene.

The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.     

SCF和APC/C的结构及功能

细胞周期蛋白依赖性蛋白激酶(cyclin dependent kinases,CDKs)是细胞周期进行的推动力,泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)通过对细胞周期蛋白(cyclin)和CDK抑制物(CDK inhibitors,CKIs)的蛋白质水解作用来实现对CDKs活性的调控。SCF(Skp1-Cul1-F-box protein)和APC/C(anaphase-promoting complex/cyclosome)这两个泛素连接酶复合物参与了很多细胞周期调节因子的泛素化作用。它们参与的蛋白质降解系统的功能失调可能导致细胞增殖紊乱、基因组不稳定和肿瘤的发生。现对这两个泛素连接酶复合物的结构以及它们在细胞周期调控和肿瘤发生机制中的作用进行综述。     

The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive

NADPH thioredoxin reductase C （NTRC） is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase （NTR） and thioredoxin （TRX） activities for the efficient reduction of 2-Cys peroxiredoxin （2-Cys PRX）. Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G- V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.