3 research outputs found
Processive Degradation of Unstructured Protein by Escherichia coli Lon Occurs via the Slow, Sequential Delivery of Multiple Scissile Sites Followed by Rapid and Synchronized Peptide Bond Cleavage Events
Processive
protein degradation is a common feature found in ATP-dependent
proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda
N protein (λN) to initiate the first kinetic analysis of the
proteolytic mechanism of this enzyme. To this end, experiments were
designed to determine the timing of three selected scissile sites
in λN approaching the proteolytic site of ELon and their subsequent
cleavages to gain insight into the mechanism by which ATP-dependent
proteases attain processivity in protein degradation. The kinetic
profile of peptide bond cleavage at different regions of λN
was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic
λN constructs were then generated as reporter substrates for
transient kinetic characterization of the ATP- versus AMPPNP-dependent
peptide bond cleavage and the delivery of the scissile sites near
the amino- versus carboxyl-terminal of the λN protein to the
proteolytic site of ELon. Collectively, our results support a mechanism
by which the cleavage of multiple peptide bonds awaits the “almost
complete” delivery of all the scissile sites in λN to
the proteolytic site in an ATP-dependent manner. Comparing the time
courses of delivery to the active site of the selected scissile sites
further implicates the existence of a preferred directionality in
the final stage of substrate delivery, which begins at the carboxyl-terminal.
The subsequent cleavage of the scissile sites in λN, however,
appears to lack a specific directionality and occurs at a much faster
rate than the substrate delivery step
The Tipper–Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA)
The
transpeptidases involved in the synthesis of the bacterial
cell wall (also known as penicillin-binding proteins, PBPs) have evolved
to bind the acyl-d-Ala-d-Ala segment of the stem
peptide of the nascent peptidoglycan for the physiologically important
cross-linking of the cell wall. The Tipper–Strominger hypothesis
stipulates that β-lactam antibiotics mimic the acyl-d-Ala-d-Ala moiety of the stem and, thus, are recognized
by the PBPs with bactericidal consequences. We document that this
mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different
classes of β-lactam antibiotics, as mimics of the acyl-d-Ala-d-Ala moiety at the allosteric site, lead to a conformational
change, across a distance of 60 Å to the active site. We directly
visualize this change using an environmentally sensitive fluorescent
probe affixed to the protein loops that frame the active site. This
conformational mobility, documented in real time, allows antibiotic
access to the active site of PBP2a. Furthermore, we document that
this allosteric trigger enables synergy between two different β-lactam
antibiotics, wherein occupancy at the allosteric site by one facilitates
occupancy by a second at the transpeptidase catalytic site, thus lowering
the minimal-inhibitory concentration. This synergy has important implications
for the mitigation of facile emergence of resistance to these antibiotics
by MRSA
Muropeptide Binding and the X‑ray Structure of the Effector Domain of the Transcriptional Regulator AmpR of <i>Pseudomonas aeruginosa</i>
A complex
link exists between cell-wall recycling/repair and the
manifestation of resistance to β-lactam antibiotics in many <i>Enterobacteriaceae</i> and <i>Pseudomonas aeruginosa</i>. This process is mediated by specific cell-wall-derived muropeptide
products. These muropeptides are internalized into the cytoplasm
and bind to the transcriptional regulator AmpR, which controls the
cytoplasmic events that lead to expression of β-lactamase, an
antibiotic-resistance determinant. The effector-binding domain (EBD)
of AmpR was purified to homogeneity. We document that the EBD exists
exclusively as a dimer, even at a concentration as low as 1 μM.
The EBD binds to the suppressor ligand UDP-<i>N</i>-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-<i>meso</i>-DAP-d-Ala-d-Ala and binds to two activator muropeptides, <i>N</i>-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-<i>N</i>-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-<i>meso</i>-DAP-d-Ala-d-Ala
and 1,6-anhydro-<i>N</i>-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-<i>meso</i>-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry.
The EBD does <i>not</i> bind to 1,6-anhydro-<i>N</i>-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-<i>meso</i>-DAP. This binding selectivity revises
the dogma in the field. The crystal structure of the EBD dimer was
solved to 2.2 Å resolution. The EBD crystallizes in a “closed”
conformation, in contrast to the “open” structure required
to bind the muropeptides. Structural issues of this ligand recognition
are addressed by molecular dynamics simulations, which reveal significant
differences among the complexes with the effector molecules