19 F-NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo-β-Lactamase.

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases (MBLs) is a growing problem. We describe the use of protein-observe F-19-NMR (PrOF NMR) to study the dynamics of the Sao Paulo MBL (SPM-1) from beta-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the alpha 3 and L3 regions, which flank the di-Zn-II active site, were selectively F-19-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile a3 and L3 regions in the binding of both inhibitors and hydrolyzed beta-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by beta-lactams and non-beta-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.