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