'Light up' protein-protein interaction through bioorthogonal incorporation of a turn-on fluorescent probe into ß-lactamase.

Fluorescent labeling of biomacromolecules to 'light up' biological events through non-invasive methods is of great importance, but is still challenging in terms of fluorophore properties and the labeling methods used. Herein, we designed and synthesized a biocompatible and conformation sensitive tetraphenylethene derivative EPB with aggregation induced emission (AIE) properties. By introducing EPB into TEM-1 ß-lactamase (TEM-1 Bla) through a two-step approach, a conformation-dependent fluorescent sensor EPB104-Bla was genetically engineered, which was applied to monitor the protein-protein interaction (PPI) with ß-lactamase inhibitor protein (BLIP). The fluorescence signal of EPB104-Bla increases by an approximately 5-fold upon binding to BLIP, indicating that EPB-104 Bla is capable of lighting up the PPI. The dissociation constant (Kd) between EPB104-Bla and BLIP was estimated to be 0.6 µM, which is consistent with that derived from the kinetic inhibition assay. This study demonstrates that genetic modification of proteins with AIE probes might open up new opportunities to develop biosensors in PPI analysis.

Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

The Ω-loop at the active site of ß-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of ß-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered ß-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the Ω-loop and to detect the binding of diverse substrates. Our results show that this engineered ß-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the Ω-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the Ω-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum ß-lactam antibiotics. The oxyimino-cephalosporin-specific fluorescence profile of our engineered ß-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled ß-lactamase.

BACKGROUND: ß-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for ß-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood. RESULTS: Here we report the crystal structure of a Class A ß-lactamase PenP from Bacillus licheniformis 749/C with fluorescein conjugated at residue 166 after E166C mutation, both in apo form (PenP-E166Cf) and in covalent complex form with cefotaxime (PenP-E166Cf-cefotaxime), to illustrate its biosensing mechanism. In the apo structure the fluorescein molecule partially occupies the antibiotic binding site and is highly dynamic. In the PenP-E166Cf-cefatoxime complex structure the binding and subsequent acylation of cefotaxime to PenP displaces fluorescein from its original location to avoid steric clash. Such displacement causes the well-folded Ω-loop to become fully flexible and the conjugated fluorescein molecule to relocate to a more solvent exposed environment, hence enhancing its fluorescence emission. Furthermore, the fully flexible Ω-loop enables the narrow-spectrum PenP enzyme to bind cefotaxime in a mode that resembles the extended-spectrum ß-lactamase. CONCLUSIONS: Our structural studies indicate the biosensing mechanism of a fluorescein-labelled ß-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of ß-lactamase-based biosensor to detect the wide spectrum of ß-lactam antibiotics. The observation of increased Ω-loop flexibility upon conjugation of fluorophore may have the potential to serve as a screening tool for novel ß-lactamase inhibitors that target the Ω-loop and not the active site.