Structural Insights into Avibactam Binding to SHV-1 and KPC-2 Enzymes

The SHV-1 and KPC-2 β-lactamases were expressed and crystallized as described previously [18 , 25 ]. As before, we used the C-terminally truncated KPC-2 β-lactamase in which the last 4 residues were removed to facilitate improved crystallization [18 , 26 ]. KPC-2 was crystallized using vapour diffusion using a sitting drop tray with a well solution comprised of 20% PEG 6000, 100 mM potassium thiocyanate, and 100 mM citrate pH 4.0; SHV-1 was crystallized using 25% PEG 6000, 100mM Tris pH 7.5, and 0.56mM Cymal-6. A crystal of SHV-1 was soaked with 50mM avibactam (AstraZeneca, Waltham, Massachusetts, USA) in mother liquor for 40 min prior to transfer to perfluoropolyether for cryo-protection before flash freezing the crystal in liquid nitrogen. KPC-2 crystals were soaked for 8 min with 5mM avibactam in mother liquor (25% PEG 6000, 100mM citrate pH 5.0) and 20% ethylene glycol prior to flash freezing. Data was collected at the Stanford Synchrotron Radiation Lightsource and processed using HKL2000[27 ] resulting in 1.42Å and 1.8Å datasets for SHV-1 and KPC-2, respectively (data collection statistics are shown in Table 1). Starting protein coordinates for SHV-1 and KPC-2 were PDB identifiers 2H5S and 3RXW, respectively; the program MOLREP was used for molecular replacement [28 ]. The structure was refined using REFMAC and COOT[29 , 30 ]. Initial refinement and subsequent density inspection indicated the presence of a covalently bound avibactam in both the SHV-1 and KPC-2 active sites. The program PRODRG[31 ] was used to obtain the topology and refinement parameter files for avibactam for subsequent inclusion of the ligand in refinement. The program PROCHECK was used for structure validation [32 ]. Final refinement statistics are listed in Table 1. The coordinates and structure factors for the KPC-2 and SHV-1 avibactam complexes were deposited with the Protein Data Bank (PDB identifiers 4ZAM and 4ZBE, respectively).