Molecular characterization of a ghrelin-l-aptamer complex

The orexigenic peptide ghrelin is involved in various and vital physiological processes such as food uptake, regulation of body weight and glucose metabolism. At Ser3 ghrelin is modified with a fatty acid. This unique posttranslational modification renders ghrelin active to bind to the growth hormone secretagogue receptor (GHSR)1a whereas deacylated ghrelin is inactive. The L-aptamer NOX-B11 was identified to specifically bind and neutralize octanoylated, i.e. active ghrelin. L-aptamers (also termed Spiegelmers) are oligonucleotide binders built from non-natural L-nucleotides that confer resistance to nucleases. Dynamic light and X-ray scattering measurements were applied to learn more about the specific recognition of a fatty acid modification by an oligonucleotide. First data revealed that upon ghrelin binding NOX-B11 undergoes a conformational change resulting in a 1:1 Ghrelin•NOX-B11 complex. Furthermore, crystals of Ghrelin•NOX-B11 were obtained and optimized by multiple cycles of micro seeding. X-ray diffraction data were collected from a single crystal to a resolution of 2.65 Å and the space group was determined to be C2. The Matthews coefficient was calculated to be 2.75 Å3Da−1, corresponding to a solvent content of approx. 60%

Crystal structure of an extracellular superoxide dismutase from Onchocerca volvulus and implications for parasite-specific drug development

Superoxide dismutases (SODs) are metalloproteins that are responsible for the dismutation of superoxide anion radicals. SODs are consequently protective against oxidative damage to cellular components. Among other protective mechanisms, the filarial parasite Onchocerca volvulus has a well developed defense system to scavenge toxic free radicals using SODs during migration and sojourning of the microfilariae and adult worms in the human body. O. volvulus is responsible for the neglected disease onchocerciasis or `river blindness'. In the present study, an extracellular Cu/Zn-SOD from O. volvulus (OvEC-SOD) was cloned, purified and crystallized to obtain structural insight into an attractive drug target with the potential to combat onchocerciasis. The recombinant OvEC-SOD forms a dimer and the protein structure was solved and refined to 1.55 Å resolution by X-ray crystallography. Interestingly, a sulfate ion supports the coordination of the conserved copper ion. The overall protein shape was verified by small-angle X-ray scattering. The enzyme shows a different surface charge distribution and different termini when compared with the homologous human SOD. A distinct hydrophobic cleft to which both protomers of the dimer contribute was utilized for a docking approach with compounds that have previously been identified as SOD inhibitors to highlight the potential for individual structure-based drug development

Isolation, crystallization and preliminary X-ray diffraction analysis of l-amino-acid oxidase from Vipera ammodytes ammodytes venom

A novel l-amino-acid oxidase was isolated from V. ammodytes ammodytes venom and crystallized. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. Preliminary diffraction data were collected to 2.6 Å resolution

Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crab Cancer pagurus

Two trypsins from the gastric fluid of the marine crab C. pagurus were purified and crystallized and X-ray data were collected to 0.97 and 3.2 Å resolution

High-resolution crystal structure and biochemical characterization of a GH11 endoxylanase from Nectria haematococca

Enzymatic degradation of vegetal biomass offers versatile procedures to improve the production of alternative fuels and other biomass-based products. Here we present the three-dimensional structure of a xylanase from Nectria haematococca (NhGH11) at 1.0 Å resolution and its functional properties. The atomic resolution structure provides details and insights about the complex hydrogen bonding network of the active site region and allowed a detailed comparison with homologous structures. Complementary biochemical studies showed that the xylanase can catalyze the hydrolysis of complex xylan into simple xylose aldopentose subunits of different lengths. NhGH11 can catalyze the efficient breakdown of beechwood xylan, xylan polysaccharide, and wheat arabinoxylan with turnover numbers of 1730.6 ± 318.1 min$^{−1}$, 1648.2 ± 249.3 min$^{−1}$ and 2410.8 ± 517.5 min$^{−1}$ respectively. NhGH11 showed maximum catalytic activity at pH 6.0 and 45 °C. The mesophilic character of NhGH11 can be explained by distinct structural features in comparison to thermophilic GH11 enzymes, including the number of hydrogen bonds, side chain interactions and number of buried water molecules. The enzymatic activity of NhGH11 is not very sensitive to metal ions and chemical reagents that are typically present in associated industrial production processes. The data we present highlights the potential of NhGH11 to be applied in industrial biomass degradation processes

Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop

AbstractThe synthetic RNA fragment 5′-CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79–97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 Å and refined to an R-value of 24.1% also represents the first X-ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments

High resolution structures of Plasmodium falciparum GST complexes provide novel insights into the dimer–tetramer transition and a novel ligand-binding site

Protection from oxidative stress and efficient redox regulation are essential for malarial parasites which have to grow and multiply rapidly in pro-oxidant rich environments. Therefore, redox active proteins currently belong to the most attractive antimalarial drug targets. The glutathione S-transferase from Plasmodium falciparum (PfGST) is a redox active protein displaying a peculiar dimer–tetramer transition that causes full enzyme-inactivation. This distinct structural feature is absent in mammalian GST isoenzyme counterparts. A flexible loop between residues 113–119 has been reported to be necessary for this tetramerization process. However, here we present structural data of a modified PfGST lacking loop 113–119 at 1.9 Å resolution. Our results clearly show that this loop is not essential for the formation of stable tetramers. Moreover we present for the first time the structures of both, the inactive and tetrameric state at 1.7 Å and the active dimeric state in complex with reduced glutathione at 2.4 Å resolution. Surprisingly, the structure of the inactive tetrameric state reveals a novel non-substrate binding-site occupied by a 2-(N-morpholino) ethane sulfonic acid (MES) molecule in each monomer. Although it is known that the PfGST has the ability to bind lipophilic anionic ligands, the location of the PfGST ligand-binding site remained unclear up to now

Structural basis to repurpose boron-based proteasome inhibitors Bortezomib and Ixazomib as β-lactamase inhibitors

β-lactamases are a major cause of rapidly emerging and spreading antibiotic resistance. Currently β-lactamase inhibitors (BLIs) in clinical use act only on Ambler Class A, C and some class D lactamases. The urgent need to identify new BLIs recently lead to FDA approval of boron-based compounds BLIs, e.g. Vaborbactam. The boron-based proteasome inhibitors Bortezomib and Ixazomib are used in cancer therapy as multiple myeloma drugs but they also bind to Ser-/Thr- proteases. In this study we show the crystal structures of the β-lactamase CTX-M-14 with covalently bound Bortezomib and Ixazomib at high resolutions of 1.3 and 1.1 Å, respectively. Ixazomib is well defined in electron density whereas Bortezomib show some disorder which corresponds to weaker inhibition efficiency observed for Ixazomib. Both inhibitors mimic the deacylation transition state of β-lactam hydrolysis, because they replace the deacylating water molecule. We further investigate differences in binding of Bortezomib/Ixazomib to CTX-M-14 and its target proteases as well as known β-lactamase drugs. Our findings can help to use Bortezomib/Ixazomib as lead compounds for development of new BLIs

A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

Many biochemical processes take place on timescales ranging from femto­seconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach for in situ crystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime