The first non-helical Aib-containing hexapeptide: The crystal structure of Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu.

The synthetic peptide Z-Gly-Aib-Gly-Aib-Gly-Aib-OtBu was crystallized from a mixture of ethyl acetate and n-hexane. The crystals belong to the centrosymmetric space group Pbca. There are three molecules in the asymmetric unit. The three molecules differ mainly in the Z-group conformation. The first Gly residue adopts a fully extended conformation, residues 2 and 3 lie in the left-handed helical region, residues 4 and 5 in the right-handed helical region, and residue 6 again in the left-handed helical region of the Ramachandran plot. There are only two of four possible intramolecular hydrogen bonds formed, namely, between Aib4 and Gly1 forming a ß-turn of type III' and between Aib6 and Gly3 forming a ß-turn of type I. The inverted molecules (by space group symmetry) lie in the regions with opposite handedness and form ß-turns of type III and I'. In contrast to all known long synthetic and naturally occurring Aib-containing peptides that fold as 310 - or α-helix, Z-(Gly-Aib)3 -OtBu folds in a quite flat structure from which only the protecting groups bulge out.

The crystal structure of Z-(Aib)10-OH at 0.65 Å resolution: three complete turns of 310-helix.

The synthetic peptide Z-(Aib)10-OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub-atomic resolution, where the bonding electron density becomes visible between the non-hydrogen atoms. Crystals belong to the centrosymmetric space group P1. Both molecules in the asymmetric unit form regular 310 -helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib-homopeptides. These other peptides are C-terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C-terminal protection group to form a hydrogen bond to the residue i-3, the sense of the helix is reversed in the last residue. Here, the C-terminal OH-groups form hydrogen bonds to the residues i-3, in part mediated by water molecules. This makes Z-(Aib)10-OH an Aib-homopeptide with three complete 310-helical turns in spite of the shorter length it has compared with Z-(Aib)11-OtBu, the only homopeptide to date with three complete turns.

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.

Z-Ala-Ile-OH, a dipeptide building block suitable for the formation of orthorhombic microtubes.

Self-assembling dipeptides have emerged in the last two decades as promising building blocks for the development of novel biomaterials. Among the various classes of dipeptides, aromatic dipeptides and especially diphenylalanine (Phe-Phe), which forms hexagonal nanotubes, have been the most extensively studied. However, aliphatic peptides or mixed aromatic-aliphatic dipeptides seem just as promising, exhibiting various structures ranging from amyloid fibrils to microtubes. Herein we report the single-crystal structure of an aliphatic dipeptide, alanine-isoleucine (Ala-Ile), C17H24N2O5, protected with a benzyloxycarbonyl (Z) group at the N-terminus. The protected dipeptide crystallizes in the orthorhombic space group P212121 and forms hollow microtubes with orthorhombic symmetry upon evaporation on glass surfaces, as shown by field emission scanning electron microscopy (FESEM). These findings provide an increased understanding of the correlation between the single-crystal structure of the peptide building block and its self-assembly mechanism, and expand the library of available building blocks for microtechnological applications.

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.

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.

Removing the C-terminal protecting group enlarges the crystal size: Z-(Gly-Aib)2-OH·H2O.

The achiral tetrapeptide monohydrate N-(benzyloxycarbonyl)glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid monohydrate, Z-Gly-Aib-Gly-Aib-OH·H2O (Z is benzyloxycarbonyl, Aib is α-aminoisobutyric acid and Gly is glycine) or C20H28N4O7·H2O, exhibits two conformations related by the symmetry operation of an inversion centre. It adopts only one of two possible intramolecular hydrogen bonds in a type I (and I') ß-turn and forms a maximum of intermolecular hydrogen bonds partly mediated by water. The space group, the molecular structure and the crystal packing differ from two already described (Gly-Aib)2 peptides which vary only in the protecting groups. This structure confirms the high structural flexibility of Gly-Aib peptides and points to a strong relationship between intermolecular hydrogen bonding and crystal quality and size.

Structure and Dynamics of a Thermostable Alcohol Dehydrogenase from the Antarctic Psychrophile Moraxella sp. TAE123.

The structure of a recombinant (His-tagged at C-terminus) alcohol dehydrogenase (MoADH) from the cold-adapted bacterium Moraxella sp. TAE123 has been refined with X-ray diffraction data extending to 1.9 Å resolution. The enzyme assumes a homo-tetrameric structure. Each subunit comprises two distinct structural domains: the catalytic domain (residues 1-150 and 288-340/345) and the nucleotide-binding domain (residues 151-287). There are two Zn2+ ions in each protein subunit. Two additional zinc ions have been found in the crystal structure between symmetry-related subunits. The structure has been compared with those of homologous enzymes from Geobacillus stearothermophilus (GsADH), Escherichia coli (EcADH), and Thermus sp. ATN1 (ThADH) that thrive in environments of diverse temperatures. Unexpectedly, MoADH has been found active from 10 to at least 53 °C and unfolds at 89 °C according to circular dichroism spectropolarimetry data. MoADH with substrate ethanol exhibits a small value of activation enthalpy ΔH ‡ of 30 kJ mol-1. Molecular dynamics simulations for single subunits of the closely homologous enzymes MoADH and GsADH performed at 280, 310, and 340 K showed enhanced wide-ranging mobility of MoADH at high temperatures and generally lower but more distinct and localized mobility for GsADH. Principal component analysis of the fluctuations of both ADHs resulted in a prominent open-close transition of the structural domains mainly at 280 K for MoADH and 340 K for GsADH. In conclusion, MoADH is a very thermostable, cold-adapted enzyme and the small value of activation enthalpy allows the enzyme to function adequately at low temperatures.

Crystal structures of Z-Gly-Aib-O-·0.5Ca2+·H2O and Z-Gly-Aib-OH.

Both deprotonated and neutral achiral title dipeptides assume similar structures of two conformations, which are related by a unit-cell inversion centre. Two mol-ecules of both conformations of the metal-free neutral dipeptide are linked by two hydrogen bonds, while two mol-ecules of both conformations of the ionized form coordinate a calcium ion in calcium(II) bis-[2-(2-{[(benz-yl-oxy)carbon-yl]amino}-acetamido)-2-methyl-propano-ate] monohydrate, 0.5Ca2+·C14H17N2O5-·0.5H2O, which lies on an inversion centre and forms a distorted octa-hedral complex with the metal ion. These CaII complexes are connected in the crystal via hydrogen bonds in the b- and c-axis directions, whereas in the a-axis direction, they stack via apolar contacts. In the metal-free crystal, namely 2-(2-{[(benz-yloxy)carbon-yl]amino}-acetamido)-2-methyl-propanoic acid, C14H18N2O5, mol-ecules are hydrogen bonded in the a- and c-axis directions, and stack in the b-axis direction via apolar contacts.

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.

Diphenylalanine in tetrahydrofuran: a highly potent candidate for the development of novel nanomaterials.

The peptide di-L-phenylalanine (FF) has emerged as a highly potent candidate for the development of novel nanomaterials. The unprotected peptide was dissolved in 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) mixed with tetrahydrofuran (THF) and single crystals of the THF monosolvate, C18H20N2O3·C4H8O, were grown by slow evaporation in a `vial-in-closed-bottle' system. THF is a molecule that can only act as a hydrogen-bond acceptor. Thus, the hydrogen-bond patterns observed in the crystal structures at 100 and 299 K are different compared to that of crystals grown from water and methanol [Mason et al. (2014). ACS Nano. 8, 1243-1253].

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.

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.

Release of Plasmodium sporozoites requires proteins with histone-fold dimerization domains.

The sporozoite, the stage of the malaria parasite transmitted by the mosquito, first develops for ∼2 weeks in an oocyst. Rupture of the oocyst capsule is required for release of sporozoites, which then transfer to the salivary gland where they are injected into a new host. Here we identify two parasite proteins that we call oocyst rupture proteins 1 (ORP1) and ORP2. These proteins have a histone-fold domain (HFD) that promotes heterodimer formation in the oocyst capsule at the time of rupture. Oocyst rupture is prevented in mutants lacking either protein. Mutational analysis confirms the HFD as essential for ORP1 and ORP2 function, and heterodimer formation was verified in vitro. These two proteins are potential targets for blocking transmission of the parasite in the mosquito.

The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

Zinc-substituted pseudoazurin solved by S/Zn-SAD phasing.

The copper(II) centre of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by zinc(II) via denaturing the protein, chelation and removal of copper and refolding the apoprotein, followed by the addition of an aqueous solution of ZnCl2. Vapour-diffusion experiments produced colourless hexagonal crystals (space group P65), which when cryocooled had unit-cell parameters a=b=49.01, c=98.08 Å. Diffraction data collected at 100 K using a copper sealed tube were phased by the weak anomalous signal of five S atoms and one Zn atom. The structure was fitted manually and refined to 1.6 Šresolution. The zinc-substituted protein exhibits similar overall geometry to the native structure with copper. Zn2+ binds more strongly to its four ligand atoms (His40 Nδ1, Cys78 Sγ, His81 Nδ1 and Met86 Sδ) and retains the tetrahedral arrangement, although the structure is less distorted than the native copper protein.

Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa.

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl2 for normal Michaelis-Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl2. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 µM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.

The achiral tetrapeptide Z-Aib-Aib-Aib-Gly-OtBu.

The title achiral peptide N-benzyloxycarbonyl-α-aminoisobutyryl-α-aminoisobutyryl-α-aminoisobutyrylglycine tert-butyl ester or Z-Aib-Aib-Aib-Gly-OtBu (Aib is α-aminoisobutyric acid, Z is benzyloxycarbonyl, Gly is glycine and OtBu indicates the tert-butyl ester), C26H40N4O7, is partly hydrated (0.075H2O) and has two different conformations which together constitute the asymmetric unit. Both molecules form incipient 310-helices. They differ in the relative orientation of the N-terminal protection group and at the C-terminus. There are two 4→1 intramolecular hydrogen bonds.

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 cobalt-substituted pseudoazurin from Alcaligenes faecalis.

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl(2). Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6(5), with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ(1), Cys78-Sγ, His81-Nδ(1), and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin.