Structural insights into the inhibition of extended-spectrum β-lactamase PER-2 by Avibactam (AVI)

K. M. PAPP-WALLACE; S. KLINKE; M. RUGGIERO; R. A. BONOMO; G. GUTKIND; P. POWER

Santo Stefano si Sessanio

Congreso; 13rd Beta-Lactamase Meeting; 2017

University of L'Aquila

Background: The PER-2 β-lactamase possesses unique structural motifs that expand the active site entrance by 2-fold vs. other class A β-lactamases. Importantly, a unique hydrogen bonding network is also present and may play a role in PER-2?s different catalytic spectrum compared to other class A β-lactamases. AVI is a reversible diazabicyclooctane (DBO) β-lactamase inhibitor (BLI) that inactivates class A and C β-lactamases. Our goal was to provide insights into the ability of AVI to inhibit PER-2 and to probe the mechanism of inhibition.Methods: Crystals of PER-2 in complex with AVI were generated by the hanging drop vapor diffusion method at 20°C. The AVI adduct was obtained by soaking the crystals of the apo-PER-2 in 7-15 mM AVI in the mother liquors for 24 h. X-ray diffraction was measured in a Bruker D8 QUEST microfocus source (Argentina) and at the Soleil synchrotron (France) under cryogenic conditions (100 K). Indexing, integration and scaling were performed with XDS, and the structure was solved by molecular replacement with Phaser. Refinement and model building were performed with Phenix, and Coot, respectively. The model was visualized with PyMol. Results: The structure of PER-2-AVI complex was refined at 2.4 Å showed 4 monomers/asymmetric unit. We observed that the W105 side chain moved >5 Å towards the active site upon the binding of AVI. The shift of W105 was accompanied by a ~4 Å shift in the T104 side chain, such that T104 now forms a new hydrogen bonding interaction with H170. Residue H170 is present in the inverted -loop of PER-2 and such that hydrogen bonding interactions are also formed with N132 and the E166 carbonyl resulting in a stable PER-2-AVI complex. Conclusion: Insights into the structures of PER-2 in complex with AVI reveal distinctive interactions of AVI with residues in the active site of this unique ESBL. The significant rearrangements and movement of residues W105 and T104 upon binding of AVI seem to be unprecedented compared to other class A enzymes.