Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species.

Orbitides are small cyclic peptides with a diverse range of therapeutic bioactivities. They are produced by many plant species, including those of the Jatropha genus. Here, the objective was to provide new structural information on orbitides to complement the growing knowledge base on orbitide sequences and activities by focusing on three Jatropha orbitides: ribifolin (1), pohlianin C (7), and jatrophidin (12). To determine three-dimensional structures, racemic crystallography, an emerging structural technique that enables rapid crystallization of biomolecules by combining equal amounts of the two enantiomers, was used. The high-resolution structure of ribifolin (0.99 Å) was elucidated from its racemate and showed it was identical to the structure crystallized from its l-enantiomer only (1.35 Å). Racemic crystallography was also used to elucidate high-resolution structures of pohlianin C (1.20 Å) and jatrophidin (1.03 Å), for which there was difficulty forming crystals without using racemic mixtures. The structures were used to interpret membrane permeability data in PAMPA and a Caco-2 cell assay, showing they had poor permeability. Overall, the results show racemic crystallography can be used to obtain high-resolution structures of orbitides and is useful when enantiopure samples are difficult to crystallize or solution structures from NMR are of low resolution.

Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase.

The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins.

The Arabidopsis B3 domain protein VERNALIZATION1 (VRN1) is involved in processes essential for development, with structural and mutational studies revealing its DNA-binding surface.

The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence-nonspecific binding, we crystallized VRN1(208-341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface.

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species.

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents, are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetrahydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculeata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous 1H and 13C NMR data are presented for these alkaloids.

Dehydration converts DsbG crystal diffraction from low to high resolution.

Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 A resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 A resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.

Thermochromism and Structure of Piperazinium Tetrachlorocuprate(II) Complexes.

The tetrachlorocuprate(II) ion can crystallize in two different structures with the piperazinium dication (pipzH(2)). Both structures contain discrete CuCl(4)(2-) species. A yellow compound (pipzH(2))[CuCl(4)].2H(2)O (1) is monoclinic (C2/c, Z = 4, a = 10.538(3) Å, b = 7.4312(5) Å, c = 17.281(4) Å, beta = 111.900(10) degrees ) and contains the CuCl(4)(2-) ion as a distorted tetrahedron. A green compound (pipzH(2))(2)[CuCl(4)].Cl(2).3H(2)O (2) is triclinic (P&onemacr;, Z = 2, a = 9.264(3) Å, b = 10.447(2) Å, c = 11.366(2) Å, alpha = 68.38 degrees, beta = 82.86(2) degrees, gamma = 83.05(2) degrees ) and contains the CuCl(4)(2-) ion with a square planar geometry. This latter compound shows thermo/photochromism, changing from green to yellow upon heating or laser irradiation.