The crystal structure of the lipoaminopeptaibol helioferin, an antibiotic peptide from Mycogone rosea.

The crystal structure of the natural nonapeptide antibiotic helioferin has been determined and refined to 0.9 Šresolution. Helioferin consists of helioferin A and B, which contain 2-(2'-aminopropyl)aminoethanol (Apae) and 2-[(2'-aminopropyl)methylamino]ethanol (Amae) at their respective alkanolamine termini. In addition, helioferin contains the unusual amino-acid residues α-aminoisobutyric acid (Aib) and (2S,4S,6S)-2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (Ahmod). The amino-terminus is capped with 2-methyl-n-1-octanoic acid (M8a). The peptide crystallizes with a 1:1 molar ratio of helioferin A and B in the monoclinic space group C2, with unit-cell parameters a = 34.711, b = 10.886, c = 17.150 Å, ß = 93.05°. The peptide backbone folds in a regular right-handed α-helical conformation, with eight intramolecular hydrogen bonds, all but one forming 5→1 interactions. The two aliphatic chains of the fatty-acyl (M8a) and the second residue (Ahmod) extend out of the α-helical structure in opposite directions and lead to a corkscrew-like shape of the peptide molecule. Halogen anions (Cl- and F-) have been co-crystallized with the peptide molecules, implying a positive charge at the aminoalcohol end of the peptide. In the tightly packed crystal the helices are linked head to tail via the anions by electrostatic, hydrogen-bond and van der Waals interactions, forming continuous helical rods. Two nonparallel rods (forming an angle of 118°) interact directly via hydrogen bonds and via the anions, forming a double layer. Successive double layers are held together only via van der Waals contacts. The helical axes of successive double layers are also related by an angle of 118°. The structure of helioferin reported here and the previously determined structure of the homologous leucinostatin A have a total straight length of about 21 Å, indicating a different membrane-modifying bioactivity from that of long-chain, amphiphilic peptaibols.

A natural, single-residue substitution yields a less active peptaibiotic: the structure of bergofungin A at atomic resolution.

Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Šresolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

The crystal structure of Z-Gly-Aib-Gly-Aib-OtBu.

The synthetic peptide Z-Gly-Aib-Gly-Aib-OtBu was dissolved in methanol and crystallized in a mixture of ethyl acetate and petroleum ether. The crystals belong to the centrosymmetric space group P4/n that is observed less than 0.3% in the Cambridge Structural Database. The first Gly residue assumes a semi-extended conformation (φ ±62°, ψ ∓131°). The right-handed peptide folds in two consecutive ß-turns of type II' and type I or an incipient 310 -helix, and the left-handed counterpart folds accordingly in the opposite configuration. In the crystal lattice, one molecule is linked to four neighbors in the ab-plane via hydrogen bonds. These bonds form a continuous network of left- and right-handed molecules. The successive ab-planes stack via apolar contacts in the c-direction. An ethyl acetate molecule is situated on and close to the fourfold axis.

The peptide Z-Aib-Aib-Aib-L-Ala-OtBu.

The title peptide, N-benzyloxycarbonyl-α-aminoisobutyryl-α-aminoisobutyryl-α-aminoisobutyryl-L-alanine tert-butyl ester or Z-Aib-Aib-Aib-L-Ala-OtBu (Aib is α-aminoisobutyric acid, Z is benzyloxycarbonyl and OtBu indicates the tert-butyl ester), C27H42N4O7, is a left-handed helix with a right-handed conformation in the fourth residue, which is the only chiral residue. There are two 4→1 intramolecular hydrogen bonds in the structure. In the lattice, molecules are hydrogen bonded to form columns along the c axis.

The crystal structure of samarosporin I at atomic resolution.

The atomic resolution structures of samarosporin I have been determined at 100 and 293 K. This is the first crystal structure of a natural 15-residue peptaibol. The amino acid sequence in samarosporin I is identical to emerimicin IV and stilbellin I. Samarosporin is a peptide antibiotic produced by the ascomycetous fungus Samarospora rostrup and belongs to peptaibol subfamily 2. The structures at both temperatures are very similar to each other adopting mainly a 310-helical and a minor fraction of α-helical conformation. The helices are significantly bent and packed in an antiparallel fashion in the centered monoclinic lattice leaving among them an approximately 10-Å channel extending along the crystallographic twofold axis. Only two ordered water molecules per peptide molecule were located in the channel. Comparisons have been carried out with crystal structures of subfamily 2 16-residue peptaibols antiamoebin and cephaibols. The repercussion of the structural analysis of samarosporin on membrane function is discussed.

Four complete turns of a curved 310-helix at atomic resolution: the crystal structure of the peptaibol trichovirin I-4A in a polar environment suggests a transition to α-helix for membrane function.

The first crystal structure of a member of peptaibol antibiotic subfamily 4, trichovirin I-4A (14 residues), has been determined by direct methods and refined at atomic resolution. The monoclinic unit cell has two molecules in the asymmetric unit. Both molecules assume a 310 right-handed helical conformation and are significantly bent. The molecules pack loosely along the crystallographic twofold axis, forming two large tunnels between symmetry-related molecules in which no ordered solvent could be located. Carbonyl O atoms which are not involved in intramolecular hydrogen bonding participate in close van der Waals interactions with apolar groups. The necessary amphipathicity for biological activity of peptaibols is not realised in the crystal structure. Hence, a structural change of trichovirin to an α-helical conformation is proposed for membrane integration and efficient water/ion transportation across the lipid bilayer.

Novel mutations in Smad proteins that inhibit signaling by the transforming growth factor beta in mammalian cells.

Smad proteins are the key effectors of the transforming growth factor beta (TGFbeta) signaling pathway in mammalian cells. The importance of Smads for human physiology is documented by the identification and characterization of mutations that are frequently found in cancer patients. In the present study we have functionally characterized such a tumorigenic mutation in Smad4 (E330A) and shown that this mutant as well as a Smad3 mutant bearing the corresponding mutation (Smad3 E239A) failed to activate transcription in response to TGFbeta stimulation because of defects in homo-and hetero-oligomerization. In the case of Smad3, the E239A mutation also abolished the phosphorylation by the TGFbeta type I receptor (ALK5). Examination of the previously published crystal structure of a Smad3/Smad4 MH2 heterotrimer [Protein Data Bank accession code, 1U7F] showed that (a) residue E239 in Smad3 participates in a dense network of intermolecular hydrogen bond and ionic interactions with other conserved polar residues such as Y237 of beta1 strand, N276 and R279 of L2 loop, and R287 of helix H1; (b) residue R287 in Smad3 is also involved in intermolecular interactions by making hydrogen and ionic bonds with Y364 in Smad3 and D493 in Smad4, an amino acid residue that is also frequently mutated in cancer patients (mutation D493H). To investigate the contribution of these interactions to Smad function and TGFbeta signaling, we replaced two of these polar residues (R287 and Y237) with a nonpolar residue (alanine) and functionally characterized the resulting Smad3 mutants. Our analysis showed that Smad3 mutant R287A was phosphorylated by the ALK5 receptor but was unable to form homo-oligomers or hetero-oligomers with Smad4 and activate transcription whereas mutation Y237A had a wild type phenotype. In summary, our present work provides a molecular basis for the functional inactivation of the TGFbeta pathway in patients bearing previously uncharacterized mutations in Smad4 as well as new information regarding the importance of conserved polar amino acids for the structure and function of the MH2 domain of Smads.

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.

Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg.

A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an alpha + beta N-terminal domain (residues 4-190) and a mainly alpha-helical C-terminal domain (residues 191-257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170.