In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.
The use of antibiotics is threatened by the emergence
and spread
of multidrug-resistant strains of bacteria. Thus, there is a need
to develop antibiotics that address new targets. In this respect,
the bacterial divisome, a multi-protein complex central to cell division,
represents a potentially attractive target. Of particular interest
is the FtsQB subcomplex that plays a decisive role in divisome assembly
and peptidoglycan biogenesis in E. coli
. Here, we report the structure-based design of
a macrocyclic covalent inhibitor derived from a periplasmic region
of FtsB that mediates its binding to FtsQ. The bioactive conformation
of this motif was stabilized by a customized cross-link resulting
in a tertiary structure mimetic with increased affinity for FtsQ.
To increase activity, a covalent handle was incorporated, providing
an inhibitor that impedes the interaction between FtsQ and FtsB irreversibly. The covalent inhibitor reduced the growth of an outer
membrane-permeable E. coli strain,
concurrent with the expected loss of FtsB localization, and also affected
the infection of zebrafish larvae by a clinical E.
coli strain. This first-in-class inhibitor of a divisome
protein–protein interaction highlights the potential of proteomimetic
molecules as inhibitors of challenging targets. In particular, the
covalent mode-of-action can serve as an inspiration for future antibiotics
that target protein–protein interactions.
The 5'-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5'-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5'-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5'-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5'-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.
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