Showing papers by "George M. Sheldrick published in 2010"

Experimental phasing with SHELXC/D/E: combining chain tracing with density modification

Abstract: The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue α-­helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a `no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap.

Labyrinthopeptins: A New Class of Carbacyclic Lantibiotics

Abstract: Lantibiotics are peptides that are ribosomally synthesized from bacteria such as staphylococci, lactobacilli, and actinomycetes. The common structural characteristic of lantibiotics is the noncanonical amino acid lanthionine (Lan, 1; Figure 1), which confers conformational stability to the peptide. The most prominent representative is nisin, which is a lipid II binder, and has been known for its use as an antimicrobial food preservative for over 40 years. The majority of studies on molecular targets and bioactivities are focused on potential applications of lantibiotics as anti-infectives. Duramycin (Moli1901) is in phase II clinical trials for the treatment of cystic fibrosis because of its ability to increase chloride transport in airway epithelium. Biosurfactant function in the life cycle of streptomycetes has been elucidated for some members such as SapB. Herein, we present the structures, the biosynthesis gene cluster, and the bioactivities of labyrinthopeptins, which are lantibiotics that contain labionin, an unprecedented carbacyclic, posttranslationally modified amino acid. The culture extracts of the novel actinomycete Actinomadura namibiensis DSM 6313 attracted our attention because of their activity against the Herpes simplex virus. Active fractions of the extracts contained a peptide that was isolated by chromatographic methods. The high-resolution ESI-FTICR mass spectrum showed a mass of 984.3333 Da for the doubly charged sodium adduct of the compound, corresponding to a neutral monoisotopic mass of 1922.6872 Da and the molecular formula C85H110N20O24S4 (Dm/m= 0.7 ppm). Amino acid analysis revealed Gly and the l-enantiomers of Ala, Thr, Leu, Asx, Cys, Phe, Glx, Trp (ratio 1:1:1:2:1:2:1:1:2). However, the total molecular mass of the detected amino acids indicated a considerable mass difference, which could not be correlated with known peptidic or lantibiotic posttranslational modifications. Resolution of the structure by H NMR spectroscopy was impeded by broad signals in parts of the spectrum. The X-ray structure at 1.0 resolution (Figure 1) enabled interpretation of the analytical data and displayed several unique structural features. In view of its labyrinthine structure, the compound was named labyrinthopeptin A2 (2). Labyrinthopeptin A2 has a globular structure that consists primarily of hydrophobic amino acids. Formally, the structure can be dissected into two nonapeptides. Each peptide bears a C-terminal Cys residue that forms a disulfide bond, which is a comparatively rare modification in lantibiotics, but is found for sublancin 168 from B. subtilis. Each nonapeptide contains a tetrapeptide (ring A) and a pentapeptide (ring B) that share a quaternary aC atom; labyrinthopeptin A rings are formed by a methylene group between the aC atoms of Lab1/ Lab10 and Lab4/Lab13 (Figure 1). A carbacyclic side-chain linkage is unprecedented in peptides and proteins. We propose the name labionin (Lab) for the corresponding amino acid (Figure 1). Labionin 3 represents an aC quaternary substituted amino acid with a subtle structural resemblance to a-aminoisobutyric acid (Aib) or isovaline (Iva), which are incorporated in fungal peptaibol-type antibiotics. The stereocenters of 3 can be assigned to (2S,4S,8R)-labionin (Lab), which is consistent with the configuration of (2S,6R)lanthionine of other lantibiotics. The formation of the 11membered ring that involves 3 forces the peptide backbone into a conformation with cis-amide bonds between Asp2– Trp3 and Thr11–Gly12, respectively (Figure 1). The presence of cis-amide bonds and the absence of a hydrogen bond between Lab1–Lab4 and Lab10–Lab13, respectively, show that the turn motif in 2 is clearly different from a b-turn motif. Subsequent identification of the biosynthetic gene cluster was performed from a cosmid library of A. namibiensis by means of degenerated primer probes, followed by sequencing [*] Dr. T. Schmiederer, Dr. K. Schneider, Dr. A. Reicke, Dr. D. Butz, Dr. S. Keller, Prof. Dr. R. D. S ssmuth Technische Universit t Berlin, Fakult t II—Institut f r Chemie Strasse des 17. Juni 124, 10623 Berlin (Germany) Fax: (+49)30-314-24205 E-mail: suessmuth@chem.tu-berlin.de Homepage: http://www2.tu-berlin.de/fb5/Suessmuth/ contact.html Dr. K. Meindl, Prof. Dr. G. M. Sheldrick Universit t G ttingen (Germany)

The Crystal Structure of Non-Modified and Bipyridine-Modified PNA Duplexes

Abstract: Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)(2), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 A, respectively. The non-modified PNA duplex adopts a P-type helical structure similar to that of previously characterized PNAs. The atomic-level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P-type helical structure, its flexibility is relatively high. For example, the base-pair rise in the bipyridine-containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π-stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl-modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.

The magic triangle goes MAD: experimental phasing with a bromine derivative

Abstract: Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

Antiarol cinnamate and africanoside, a cinnamoyl triterpene and a hydroperoxy-cardenolide from the stem bark of Antiaris africana.

Abstract: From the methanol extract of the stem bark of the African tree Antiaris africana Engler, two new bioactive metabolites were isolated, namely, the α-amyrin derivative 1β,11α-dihydroxy-3β-cinnamoyl-α-amyrin (antiarol cinnamate, 1) and a cardiac glycoside, 3β-O-(α-L-rhamnopyranosyl)-14β-hydroperoxy-5β-hydroxy-19-oxo-17β-card-20(22)-enolide (africanoside, 2a), together with the known compounds β-amyrin and its acetate, β-sitosterol and its 3-O-β-D-glucopyranoside, friedelin, ursolic and oleanolic acid, 19-norperiplogenin, strophanthidol, strophanthidinic acid, periplogenin (3a), 3-epiperiplogenin, strophanthidin (3b) and 3,3'-dimethoxy-4'-O-β-D-xylopyronosyl-ellagic acid. Their structures were established on the basis of their spectroscopic data and by chemical methods, while 3a was additionally confirmed by X-ray crystal structure analysis. The aglycone moiety possessing a hydroperoxy group was found for the first time in cardenolides. Compounds 1 and 2a showed no activity against bacteria, fungi, and microalgae; however, the crude extract exhibited a high toxicity against Artemia salina and a selective antitumor activity against human tumor cell lines. Africanoside (2a) effected a concentration-dependent inhibition of tumor cell growth with a mean IC(50) value of 5.3 nM.

Structure analysis of endosialidase NF at 0.98 A resolution.

Abstract: Endosialidase NF (endoNF) is a bacteriophage-derived endosialidase that specifically degrades alpha-2,8-linked polysialic acid. The structure of a new crystal form of endoNF in complex with sialic acid has been refined at 0.98 A resolution. The 210 kDa homotrimeric multi-domain enzyme displays outstanding stability and resistance to SDS. Even at atomic resolution, only a minor fraction of side chains possess alternative conformations. However, multiple conformations of an active-site residue imply that it has an important catalytic function in the cleavage mechanism of polysialic acid.

The thermodynamic influence of trapped water molecules on a protein-ligand interaction

Abstract: Water molecules doing time: Atomic-resolution crystal structures of the PPIase domain of cyclophilin G, alone and in complex with cyclosporin A, and together with MD simulations and calorimetry, reveal how trapped water molecules influence the thermodynamic profile of a protein-ligand interaction.