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

Abstract

Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe ¹⁹ F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-Zn^II active site, were selectively ¹⁹ F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-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.

Keywords: NMR spectroscopy; São Paulo metallo-β-lactamase; antibiotic resistance; protein structures; β-lactamases.