Hana McFeeters

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.

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.

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.

A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

The emerging importance of Peptidyl-tRNA hydrolase (Pth) enzymes necessitates the need for a widely applicable functional assay to further studies of this important enzyme family. Previously reported methods for monitoring Pth function suffer from limitations of cost, time, substrate availability, and application compatibility. Herein we present a new method for the rapid and precise characterization of Pth activity. The method is applicable for use with specific or bulk peptidyl-tRNA, any Pth enzyme, and a range of reaction conditions including solvent additives. The method also allows for semi-automated quantitative assessment of peptidyl-tRNA cleavage. No specialized equipment, harmful reagents, or time-consuming techniques are required. We use the new method to characterize Pth activity, determine enzyme kinetic parameters, screen for inhibitors, and determine inhibitory parameters.

Current Methods for Analysis of Enzymatic Peptidyl-tRNA Hydrolysis

Understanding peptidyl-tRNA and the enzymes responsible for recycling them has come from the ability to detect and quantify enzymatic peptidyl-tRNA hydrolysis. The methods available to study removal of peptides from tRNA have evolved considerably. Radioactive [14C] amino acids were first implemented to monitor cleavage of the peptide-nucleotide ester bond of uniform peptidyl-tRNA substrates. Later, Northern blots with radiolabeled oligonucleotide probes were used to observe cleavage of specific peptidyl-tRNAs or individual tRNA from bulk peptidyl-tRNA populations. Finally, the use of fluorescently labeled amino acids was introduced, which could be coupled to anisotropy or PAGE readouts. Here we review the methods for quantification and analysis of enzymatic peptidyl-tRNA hydrolysis and summarize their inherent advantages and disadvantages.

Small Molecule Docking Supports Broad and Narrow Spectrum Potential for the Inhibition of the Novel Antibiotic Target Bacterial Pth1

Peptidyl-tRNA hydrolases (Pths) play ancillary yet essential roles in protein biosynthesis by recycling peptidyl-tRNA. In E. coli, inhibition of bacterial Pth1 leads to accumulation of peptidyl-tRNA, depletion of aminoacyl-tRNA, and cell death. Eukaryotes have multiple Pths and Pth1 knock out was shown to have no effect on viability in yeast. Thereby, bacterial Pth1 is a promising target for novel antibiotic development. With the abundance of Pth1 structural data, molecular docking was used for virtual screening of existing, commercially available antibiotics to map potential interactions with Pth enzymes. Overall, 83 compounds were docked to eight different bacterial Pth1 and three different Pth2 structures. A variety of compounds demonstrated favorable docking with Pths. Whereas, some compounds interacted favorably with all Pths (potential broad spectrum inhibition), more selective interactions were observed for Pth1 or Pth2 and even specificity for individual Pth1s. While the correlation between computational docking and experimentation still remains unknown, these findings support broad spectrum inhibition, but also point to the possibility of narrow spectrum Pth1 inhibition. Also suggested is that Pth1 can be distinguished from Pth2 by small molecule inhibitors. The findings support continued development of Pth1 as an antibiotic target.

Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus

Peptidyl-tRNA hydrolase 1 cleaves the ester bond of peptidyl-tRNA thereby recycling peptidyl-tRNAs generated from premature termination of translation and expression of minigenes and short ORFs. Bacterial Pth1 is essential, highly conserved, and has no essential eukaryotic homolog making it a good target for antibacterial development. Herein we describe the cloning of pth1 gene from Bacillus cereus as an N-terminal hexahistidine fusion protein. Solubility was optimized for overexpression in Escherichia coli. Purity greater than 95% was achieved in one chromatography step. Yields greater than 12mg of purified Pth1 per liter of minimal media were achieved and buffer conditions for long-term solubility were determined. Enzymatic activity of Pth1 from B. cereus was confirmed and quantification of Michaelis-Menten parameters reported.

Bacterial production of site specific 13 C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.