Robert L. McFeeters

Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78

The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

Emerging structural explanations of ionotropic glutamate receptor function

High‐resolution studies of ionotropic glutamate receptor (iGluR) extracellular domains are beginning to bridge the gap between structure and function. Crystal structures have defined the ligand binding pocket well beyond what was suggested by mutational analysis and homology models alone, providing initial suggestions about the mechanisms of channel gating and desensitization. NMR‐derived backbone dynamics and molecular dynamics simulations have added further insights into the role of protein dynamics in receptor function. As a whole, the current knowledge of iGluR structure in conjunction with new advances in the understanding of K+ channels provides a vastly improved understanding of iGluR function. This review focuses on structural and dynamic studies of the extracellular ligand binding domain of iGluRs and the pore region of K+ channels that have contributed to mechanistic insights into the processes of iGluR gating and desensitization.—McFeeters, R. L., Oswald, R. E. Emerging structural explanations of ionotropic glutamate receptor function.

Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa

The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6(1)22 with unit-cell parameters a=b=63.62, c=155.20 Å, α=β=90, γ=120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da(-1). The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.

Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations

Thyme (Thymus vulgaris L.) is a commonly used flavoring agent and medicinal herb. Several chemotypes of thyme, based on essential oil compositions, have been established, including (1) linalool; (2) borneol; (3) geraniol; (4) sabinene hydrate; (5) thymol; (6) carvacrol, as well as a number of multiple-component chemotypes. In this work, two different T. vulgaris essential oils were obtained from France and two were obtained from Serbia. The chemical compositions were determined using gas chromatography–mass spectrometry. In addition, chiral gas chromatography was used to determine the enantiomeric compositions of several monoterpenoid components. The T. vulgaris oil from Nyons, France was of the linalool chemotype (linalool, 76.2%; linalyl acetate, 14.3%); the oil sample from Jablanicki, Serbia was of the geraniol chemotype (geraniol, 59.8%; geranyl acetate, 16.7%); the sample from Pomoravje District, Serbia was of the sabinene hydrate chemotype (cis-sabinene hydrate, 30.8%; trans-sabinene hydrate, 5.0%); and the essential oil from Richerenches, France was of the thymol chemotype (thymol, 47.1%; p-cymene, 20.1%). A cluster analysis based on the compositions of these essential oils as well as 81 additional T. vulgaris essential oils reported in the literature revealed 20 different chemotypes. This work represents the first chiral analysis of T. vulgaris monoterpenoids and a comprehensive description of the different chemotypes of T. vulgaris.

Thermal coefficients of the methyl groups within ubiquitin.

Physiological processes such as protein folding and molecular recognition are intricately linked to their dynamic signature, which is reflected in their thermal coefficient. In addition, the local conformational entropy is directly related to the degrees of freedom, which each residue possesses within its conformational space. Therefore, the temperature dependence of the local conformational entropy may provide insight into understanding how local dynamics may affect the stability of proteins. Here, we analyze the temperature dependence of internal methyl group dynamics derived from the cross‐correlated relaxation between dipolar couplings of two CH bonds within ubiquitin. Spanning a temperature range from 275 to 308 K, internal methyl group dynamics tend to increase with increasing temperature, which translates to a general increase in local conformational entropy. With this data measured over multiple temperatures, the thermal coefficient of the methyl group order parameter, the characteristic thermal coefficient, and the local heat capacity were obtained. By analyzing the distribution of methyl group thermal coefficients within ubiquitin, we found that the N‐terminal region has relatively high thermostability. These results indicate that methyl groups contribute quite appreciably to the total heat capacity of ubiquitin through the regulation of local conformational entropy.

Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

Recombinant production, crystallization and X‐ray crystallographic structure determination of peptidyl‐tRNA hydrolase from Salmonella typhimurium

Peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacterium Salmonella typhimurium has been cloned, expressed in Escherichia coli and crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 M citric acid, 0.05 M bis-tris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P2₁2₁2₁ with unit-cell parameters a=62.1, b=64.9, c=110.5 Å, α=β=γ=90°. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å3 Da(-1). The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.

Scytovirin Engineering Improves Carbohydrate Affinity and HIV-1 Entry Inhibition

Scytovirin, a cyanobacterium derived carbohydrate binding protein, acts as a potent HIV-1 entry inhibitor and could hold promise as a potential topical microbicide. Viral specificity is achieved as Scytovirin recognizes carbohydrate moieties rarely found in the extracellular matrix, but which are abundant on viral proteins. With the goal to improve the anti-viral capacity of Scytovirin, we here analyze the factors contributing to the Scytovirin anti-viral effect. We show that aromatic substitutions in the lower affinity C-terminal domain of Scytovirin lead to tighter carbohydrate binding. Several other mutations or an addition to the N-terminal abolish carbohydrate binding and abrogate the antiviral effect. Moreover, the increased binding affinity translates directly to improved antiviral efficacy. These studies improve our understanding of the Scytovirin:carbohydrate interaction and provide a blueprint for additional targeted mutations to advance Scytovirin as an entry inhibitor.

Chemotypic Characterization and Biological Activity of Rosmarinus officinalis

Rosemary (Rosmarinus officinalis L.) is a popular herb in cooking, traditional healing, and aromatherapy. The essential oils of R. officinalis were obtained from plants growing in Victoria (Australia), Alabama (USA), Western Cape (South Africa), Kenya, Nepal, and Yemen. Chemical compositions of the rosemary oils were analyzed by gas chromatography-mass spectrometry as well as chiral gas chromatography. The oils were dominated by (+)-α-pinene (13.5%–37.7%), 1,8-cineole (16.1%–29.3%), (+)-verbenone (0.8%–16.9%), (−)-borneol (2.1%–6.9%), (−)-camphor (0.7%–7.0%), and racemic limonene (1.6%–4.4%). Hierarchical cluster analysis, based on the compositions of these essential oils in addition to 72 compositions reported in the literature, revealed at least five different chemotypes of rosemary oil. Antifungal, cytotoxicity, xanthine oxidase inhibitory, and tyrosinase inhibitory activity screenings were carried out, but showed only marginal activities.

α-Pyrone derivatives, tetra/hexahydroxanthones, and cyclodepsipeptides from two freshwater fungi

Eighteen (1-18) and seven (1, 4, 6-8, 17 and 18) compounds were isolated from organic extracts of axenic cultures of two freshwater fungi Clohesyomyces sp. and Clohesyomyces aquaticus (Dothideomycetes, Ascomycota), respectively. Compounds 1-12 belong to the α-pyrone class of natural products, compounds 13 and 14 were tetrahydroxanthones, compounds 15 and 16 were hexahydroxanthones, while compounds 17 and 18 were cyclodepsipeptides. The structures were elucidated using a set of spectroscopic and spectrometric techniques. The absolute configurations of compounds 2, 3, 6, and 7 were assigned via a modified Moshers ester method using 1H NMR data. The relative configurations of compounds 14-16 were determined through NOE data. Compounds 1, 2, 6, 8, 13, 14, and 15 were found to inhibit the essential enzyme bacterial peptidyl-tRNA hydrolase (Pth1), with (13; secalonic acid A) being the most potent. Compounds 1 and 4-18 were also evaluated for antimicrobial activity against an array of bacteria and fungi but were found to be inactive.