PubMed · 13118246
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G C WELLS, C BABCOCK. 1953. Epidermal protease.. https://doi.org/10.1038/jid.1953.122
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Protein-based antibacterials such as bacteriophage endolysins offer a targeted therapeutic strategy against Gram-positive pathogens. However, prioritizing the most effective candidates from the large sequence diversity available remains a significant challenge. Here we present a standardized computational-experimental benchmarking framework that evaluates seven phage-derived endolysin variants (E1, E2, E3, E7, E10, E12, and E15) identified from Bacillus genomes. We combined molecular docking and residue-level interaction mapping against muramyl dipeptide (MDP), a minimal conserved peptidoglycan motif, with 1000-ns molecular dynamics simulations, MM/PBSA binding free-energy estimation, and matched functional inhibition assays against Staphylococcus aureus and Micrococcus luteus. Computational analyses revealed generally favorable MDP recognition across variants, albeit with notable differences in contact patterns and complex stability profiles. Experimental screening identified E2 as the most potent antibacterial agent against both species, while E7 and E1 performed strongly in selected computational metrics. Integrated analysis showed only modest correlations between computational descriptors of fragment recognition/stability and observed antibacterial performance. This study establishes a practical comparative benchmarking platform for endolysin candidate prioritization, nominates E2 and E7 as promising candidates for further development, and highlights E1 as a potential structural scaffold for rational engineering, while explicitly demonstrating both the utility and the current limitations of using minimal peptidoglycan fragments as proxies for full cell-wall recognition in lysin benchmarking.
Isoprenylcysteine carboxyl methyltransferase (Icmt) catalyzes the methylation of the C-terminal prenylcysteine found on prenylated proteins. Numerous studies have shown that the methylation step is important for the correct localization and function of many prenylated proteins, most notably GTPases in the Ras superfamily. We recently reported identification of a small molecule derived from an indole core as a potent, cell-active inhibitor of Icmt whose potency was increased upon preincubation with the enzyme [Winter-Vann, A. M., Baron, R. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102 (12), 4336-41]. In the study presented here, we performed a kinetic characterization of this time-dependent inhibition of Icmt by 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). These analyses revealed that cysmethynil is a competitive inhibitor with respect to the isoprenylated cysteine substrate and a noncompetitive inhibitor with respect to AdoMet, the methyl donor in the reaction. The Ki of cysmethynil for Icmt, which represents the dissociation constant of the initial complex with the enzyme, was 2.39 +/- 0.02 microM, and the Ki*, which is the overall dissociation constant of the inhibitor for the final complex, was 0.14 +/- 0.01 microM. The first-order rate constant for the conversion of the initial enzyme-inhibitor complex to the final high-affinity complex was 0.87 +/- 0.06 min-1, and that for the reverse process was 0.053 +/- 0.003 min-1; the latter rate constant corresponds to a half-life for the high-affinity complex of 15 min. Structure-activity relationships of a number of closely related indole compounds revealed that the hydrophobicity of the substituent on the nitrogen of the indole core was responsible for the manifestation of time-dependent inhibition. These findings markedly enhance our understanding of the mechanism of inhibition of Icmt by this indole class of compounds and should facilitate ongoing efforts to assess the potential of targeting this enzyme in anticancer drug design.
Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.