2 research outputs found
General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria
The
chemical features that impact small-molecule permeability across
bacterial membranes are poorly understood, and the resulting lack
of tools to predict permeability presents a major obstacle to the
discovery and development of novel antibiotics. Antibacterials are
known to have vastly different structural and physicochemical properties
compared to nonantiinfective drugs, as illustrated herein by principal
component analysis (PCA). To understand how these properties influence
bacterial permeability, we have developed a systematic approach to
evaluate the penetration of diverse compounds into bacteria with distinct
cellular envelopes. Intracellular compound accumulation is quantitated
using LC-MS/MS, then PCA and Pearson pairwise correlations are used
to identify structural and physicochemical parameters that correlate
with accumulation. An initial study using 10 sulfonyladenosines in <i>Escherichia coli</i>, <i>Bacillus subtilis</i>, and <i>Mycobacterium smegmatis</i> has identified nonobvious correlations
between chemical structure and permeability that differ among the
various bacteria. Effects of cotreatment with efflux pump inhibitors
were also investigated. This sets the stage for use of this platform
in larger prospective analyses of diverse chemotypes to identify global
relationships between chemical structure and bacterial permeability
that would enable the development of predictive tools to accelerate
antibiotic drug discovery
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Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition
Non-ribosomal peptides play a critical role in the clinic
as therapeutic
agents. To access more chemically diverse therapeutics, non-ribosomal
peptide synthetases (NRPSs) have been targeted for engineering through
combinatorial biosynthesis; however, this has been met with limited
success in part due to the lack of proper protein–protein interactions
between non-cognate proteins. Herein, we report our use of chemical
biology to enable X-ray crystallography, molecular dynamics (MD) simulations,
and biochemical studies to elucidate binding specificities between
peptidyl carrier proteins (PCPs) and adenylation (A) domains. Specifically,
we determined X-ray crystal structures of a type II PCP crosslinked
to its cognate A domain, PigG and PigI, and of PigG crosslinked to
a non-cognate PigI homologue, PltF. The crosslinked PCP-A domain structures
possess large protein–protein interfaces that predominantly
feature hydrophobic interactions, with specific electrostatic interactions
that orient the substrate for active site delivery. MD simulations
of the PCP-A domain complexes and unbound PCP structures provide a
dynamical evaluation of the transient interactions formed at PCP-A
domain interfaces, which confirm the previously hypothesized role
of a PCP loop as a crucial recognition element. Finally, we demonstrate
that the interfacial interactions at the PCP loop 1 region can be
modified to control PCP binding specificity through gain-of-function
mutations. This work suggests that loop conformational preferences
and dynamism account for improved shape complementary in the PCP-A
domain interactions. Ultimately, these studies show how crystallographic,
biochemical, and computational methods can be used to rationally re-engineer
NRPSs for non-cognate interactions