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

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

Santo Stefano di Sessanio, L?aquila.

Congreso; 13th beta-Lactamase Meeting; 2017

Background: The PER-2 β-lactamase possesses unique structuralmotifs that expand the active site entrance by 2-fold vs. other class A β-lactamases. Importantly, a unique hydrogenbonding network is also present and may play a role in PER-2?s differentcatalytic spectrum compared to other class A β-lactamases. AVI is a reversible diazabicyclooctane(DBO) β-lactamase inhibitor (BLI) that inactivates class Aand C β-lactamases. Our goal was to provide insights into the ability of AVI toinhibit PER-2 and to probe the mechanism of inhibition.Methods: Crystals of PER-2 in complex with AVI were generated by thehanging drop vapor diffusion method at 20°C. The AVI adduct was obtained bysoaking the crystals of the apo-PER-2 in 7-15 mM AVI in the mother liquors for24 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 wereperformed with XDS, and the structure was solved by molecular replacement withPhaser. 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 refinedat 2.4 Å showed 4 monomers/asymmetric unit. We observed that the W105 sidechain moved >5 Å towards the active site upon the binding of AVI. The shiftof W105 was accompanied by a ~4 Å shift in the T104 side chain, such that T104now forms a new hydrogen bonding interaction with H170. Residue H170 is presentin the inverted W-loop of PER-2 and such that hydrogen bondinginteractions are also formed with N132 and the E166 carbonyl resulting in astable PER-2-AVI complex.Conclusion: Insights into the structures of PER-2 incomplex with AVI reveal distinctive interactions of AVI with residues in theactive site of this unique ESBL. The significant rearrangements and movement ofresidues W105 and T104 upon binding of AVI seem to be unprecedented compared toother class A enzymes.