Laszlo Vertesy

Crystal structure determination, refinement and the molecular model of the α-amylase inhibitor Hoe-467A

The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.

Labyrinthopeptins: A New Class of Carbacyclic Lantibiotics

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)

Structure of the lipopeptide antibiotic tsushimycin.

The amphomycin derivative tsushimycin has been crystallized and its structure determined at 1.0 A resolution. The asymmetric unit contains 12 molecules and with 1300 independent atoms this structure is one of the largest solved using ab initio direct methods. The antibiotic is comprised of a cyclodecapeptide core, an exocyclic amino acid and a fatty-acid residue. Its backbone adopts a saddle-like conformation that is stabilized by a Ca2+ ion bound within the peptide ring and accounts for the Ca2+-dependence of this antibiotic class. Additional Ca2+ ions link the antibiotic molecules to dimers that enclose an empty space resembling a binding cleft. The dimers possess a large hydrophobic surface capable of interacting with the bacterial cell membrane. The antibiotic daptomycin may exhibit a similar conformation, as the amino-acid sequence is conserved at positions involved in Ca2+ binding.

Structures of glycopeptide antibiotics with peptides that model bacterial cell-wall precursors

The vancomycin-related antibiotics balhimycin and degluco-balhimycin have been crystallized in complexes with di-, tri- and pentapeptides that emulate bacterial cell-wall precursors, and four structures determined at atomic resolution (<1 A). In addition to the features expected from previous structural and spectroscopic studies, two new motifs were observed that may prove important in the design of antibiotics modified to overcome bacterial resistance. A changed binding mode was found in two dipeptide complexes, and a new type of face-to-face oligomerization (in addition to the well-established back-to-back dimerization) was seen when the model peptide reaches a critical fraction of the size of the cell-wall precursor pentapeptide. The extensive interactions involving both antibiotic and peptide molecules in this interface should appreciably enhance the kinetic and thermodynamic stability of the complexes. In the pentapeptide complex, the relative positions of the peptides are close to those required for d-Ala elimination, so this structure may provide a realistic model for the prevention of the enzyme-catalyzed cell-wall crosslinking by antibiotic binding.

Structure of Balhimycin and its Complex with Solvent Molecules

Balhimycin is a naturally occurring glycopeptide antibiotic, related to vancomycin which acts by binding nascent bacterial cell-wall peptide ending in the sequence D-Ala-D-Ala. Crystals of balhimycin are monoclinic, space group P21, a = 20.48 (10), b = 43.93 (21), c = 27.76 (14) A, beta = 100.5 (5) degrees with four independent antibiotic molecules, three molecules of 2-methyl-2,4-pentanediol, two citrate ions, three acetate ions and 127.5 water molecules in the asymmetric unit. With an asymmetric unit larger than those of the smallest proteins and a solvent content of about 32%, the crystals have similar diffraction properties to those of small proteins. 27387 unique reflections were collected using synchrotron radiation. The structure was solved by a standard protein technique, the molecular-replacement method, using ureido-balhimycin as search model. The anisotropic refinement against all F2 data between 0.96 and 45 A converged to a conventional R value of 11.27% with R1= Sigma||Fo|-|Fc||/Sigma|Fo| for the 24623 data with I > 2sigma(I) and 12.58% for all 27387 data. The four monomers possess fairly similar conformations (r.m.s. deviation 0.7 A). Two antibiotic molecules form a tight dimer with antiparallel hydrogen bonds between the peptide backbone as well as between the vancosamine residues and the peptide backbone. In each of the two dimers, one binding pocket is occupied by a citrate ion and the other by an acetate ion. The dimer units are linked in the crystal by hydrogen bonds to form infinite chains.

Structure of the α-amylase inhibitor tendamistat at 0.93 Å

The crystal structure of the proteinaceous alpha-amylase inhibitor tendamistat has been determined at 100 K to a resolution of 0.93 A. The final R factor for all reflections with F > 4sigma(F) is 9.26%. The mean coordinate error for fully occupied protein atoms as derived from full-matrix inversion is 0.018 A. An extended network of multiple discrete conformations has been identified on the side of tendamistat that binds to the target molecule. Most notably, residue Tyr15, which interacts with the glycine-rich loop characteristic of mammalian amylases, and a cluster of amino-acid side chains surrounding it are found in two well defined conformations. The flexibility observed in this crystal structure together with information about residues fixed by lattice contacts in the crystal but found to be mobile in a previous NMR study supports a model in which most of the residues involved in binding are not fixed in the free form of the inhibitor, suggesting an induced-fit type of binding.

The Tendamistat Expression-Secretion System: Synthesis of Proinsulin Fusion Proteins with Streptomyces Lividans

Tendamistat (HOE 467), a potent inhibitor of the human pancreatic α-amylase, is an acidic protein of 74 amino acids (Vertesy et al., 1984). The inhibitor gene was cloned from an amplified genomic sequence of an over-producing strain of Streptomyces tendae and further characterized. By expression of the gene in the heterologous host Streptomyces lividans we demonstrated that secretion of this protein was mediated by a signal peptide dependent mechanism (Koller & Ries, 1989). We have also evaluated the tendamistat-based secretion system for a number of foreign proteins, for example interleukin II and proinsulin (Bender et al., 1990, Koller et al., 1989). Our main interest focussed on the formation of disulphide bonds, stability and activity of secreted proteins.