3 research outputs found
Identification and Co-complex Structure of a New <i>S. pyogenes</i> SpeB Small Molecule Inhibitor
The secreted <i>Streptococcus
pyogenes</i> cysteine protease
SpeB is implicated in host immune system evasion and bacterial virulence.
We present a small molecule inhibitor of SpeB <b>2477</b> identified
from a high-throughput screen based on the hydrolysis of a fluorogenic
peptide substrate Ac-AIK-AMC. <b>2477</b> inhibits other SpeB-related
proteases but not human caspase-3, suggesting that the molecule targets
proteases with the papain-like structural fold. A 1.59 Å X-ray
crystal structure of <b>2477</b> bound to the SpeB active site
reveals the mechanism of inhibition and the essential constituents
of <b>2477</b> necessary for binding. An assessment against
a panel of <b>2477</b> derivatives confirms our structural findings
and shows that a carbamate and nitrile on <b>2477</b> are required
for SpeB inhibition, as these moieties provide an extensive network
of electrostatic and hydrogen-bonding interactions with SpeB active
site residues. Surprisingly, despite <b>2477</b> having a reduced
inhibitory potential against papain, the majority of <b>2477</b>-related compounds inhibit papain to a much greater and broader extent
than SpeB. These findings indicate that SpeB is more stringently selective
than papain for this panel of small molecule inhibitors. On the basis
of our structural and biochemical characterization, we propose modifications
to <b>2477</b> for subsequent rounds of inhibitor design that
will impart specificity to SpeB over other papain-like proteases,
including alterations of the compound to exploit the differences in
CA protease active site pocket sizes and electrostatics
Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass
The
lysis and extraction of soluble bacterial proteins from cells is a
common practice for proteomics analyses, but insoluble bacterial biomasses
are often left behind. Here, we show that with triflic acid treatment,
the insoluble bacterial biomass of Gram<sup>–</sup> and Gram<sup>+</sup> bacteria can be rendered soluble. We use LC-MS/MS shotgun
proteomics to show that bacterial proteins in the soluble and insoluble
postlysis fractions differ significantly. Additionally, in the case
of Gram<sup>–</sup> Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated
proteins. Finally, we apply triflic acid to a human microbiome sample
to show that this treatment is robust and enables the identification
of a new, complementary subset of proteins from a complex microbial
mixture
Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass
The
lysis and extraction of soluble bacterial proteins from cells is a
common practice for proteomics analyses, but insoluble bacterial biomasses
are often left behind. Here, we show that with triflic acid treatment,
the insoluble bacterial biomass of Gram<sup>–</sup> and Gram<sup>+</sup> bacteria can be rendered soluble. We use LC-MS/MS shotgun
proteomics to show that bacterial proteins in the soluble and insoluble
postlysis fractions differ significantly. Additionally, in the case
of Gram<sup>–</sup> Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated
proteins. Finally, we apply triflic acid to a human microbiome sample
to show that this treatment is robust and enables the identification
of a new, complementary subset of proteins from a complex microbial
mixture