X-Ray structure of PER-2 beta-lactamase: insights into the role of R220 in the interaction with beta-lactams and beta-lactamase inhibitors

M. RUGGIERO; E. SAUVAGE; R. HERMAN; F.SAPUNARIC; M. GALLENI; G. GUTKIND; P. CHARLIER; P. POWER

Ciudad Autónoma de Buenos Aires

Congreso; XIII Congreso Argentino de Microbiología; 2013

Asociación Argentina de Microbiología

Background: The X-ray structure of PER-1 has been solved, and the role of different residues has also been studied for this variant. The structure of PER-1 revealed singular features, such as the presence of a different fold in the omega-loop, and the insertion of four residues generating a new fold at the edge of the strand B3 generating a broader catalytic cleft that could easily accommodate bulkier substrates like the oxyimino-cephalosporins (OC) cefotaxime and ceftazidime. As we recently observed, mutations at R220 appear to be somewhat equivalent to R244 in inhibitor-resistant (IRT) TEM variants, having an impact on the inhibition susceptibility of PER-2 as well as towards the substrate profile. Objectives: In this study, we determined the X-ray structure of PER-2 at 2.10 Å, and evaluated the possible role of R220 in the structure and activity towards beta-lactams and mechanism-based beta-lactamase inhibitors. Methods: PER-2 was purified to homogeneity by conventional chromatography techniques. Crystals of pure PER-2 were obtained by hanging drop vapor diffusion at 20°C. X-Ray diffraction was carried out at Soleil synchrotron (Paris, France) under cryogenic conditions (100K). Indexing/integrationand scaling of the intensity data were performed with XDS and XSCALE, respectively, and structure obtained by molecular replacement and refinement with REFMAC5, TLS, and Coot. Simulated models of PER-2 andderived mutants in combination with beta-lactams and beta-lactamase inhibitors were obtained with Coot and PyMol. Results: The structure of PER-2 was refined to 2.10 Å (PDB entry: 3znw (HPUB); Rfree: 24.2%; Ramachandran s favored residues: 96.7%). In PER-2, B3 strand is shifted up to ca. 5 Å towards the catalytic cleft in comparison with other class A beta-lactamases, creating an apparently more favorable environment for stabilization of molecules like the oxyimino-cephalosporinsthrough hydrogen bonds. This strand seems to be also stabilized by hydrogen bonds between T237 and R220. According to simulated models of wild-type and R220-mutant PER-2 beta-lactamases, the environment of residues at position 220 might have influence on both the inhibition by mechanismbased inhibitors and also the activity towards several compounds. Previously determined kinetic parameters support this behavior, especially for some substrates, for which increase Km values are accompanied by decrease in kcatvalues, yielding kcat/Km values up to 10-fold lower. Conclusions: The structural evidences suggest that the interactions of different beta-lactam molecules with the observed hydrogen-bond networks associated with the reaction coordinate residues are stabilized by specific amino acid residues, being R220 an important residue for the activity towardcephalosporins and probably the mechanism-based inhibitors. The overall stability of the R220 mutants also suggests that these variants could arise during in vivo therapies with these drugs.