19F‐NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo‐β‐Lactamase
نویسندگان
چکیده
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-ZnII 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.
منابع مشابه
Studying the active-site loop movement of the São Paolo metallo-β-lactamase-1† †Electronic supplementary information (ESI) available: Procedures for protein expression and purification, 19F-labelling, crystallisation, data collection, and structure determination, table of crystallographic data, table of crystallographic parameters and refinement statistics, figures showing binding mode and distances, procedures for mass spectrometry measurements, differential scanning fluorimetry measurements, stopped-flow measurements and other kinetics measurements. See DOI: 10.1039/c4sc01752h Click here for additional data file.
Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibiotics. We report biophysical and kinetic studies on the São Paulo MBL (SPM-1), which reveal its Zn(ii) ion usage and mechanism as characteristic of the clinically important di-Zn(ii) dependent B1 MBL subfamily. Biophysical analyses employing crystallography, dynamic 19F NMR and ion mobility mass spectrometry, howev...
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