What is it about?
Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe 19F-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 19F-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.
Featured Image
Why is it important?
The results are of interest for identifying novel inhibitor scaffolds for SPM-1 and other MBLs, including the design of non-β-lactam inhibitors that are not susceptible to β-lactamase hydrolysis, and/or β-lactams or β-lactam analogues that give hydrolyzed products that inhibit MBLs. Whist 13C/15N labeling is often powerful for studying ligand binding, it is relatively expensive and time consuming. In contrast, our results clearly illustrate the utility of PrOF NMR for studying protein–ligand interactions in solution, detecting metal chelation, and revealing subtle differences in binding modes.
Perspectives
From a catalytic perspective, the results are important because they inform on the nature of the SPM-1-product complexes in solution (the first such studies on MBLs). They reveal that the hydrolyzed β-amino acid products bind in a manner involving the L3, but not the α3, loop. The difference in binding observed for the initially formed (5R), and subsequently formed (5S), epimers of the penicilloic acid products reveals the utility of PrOF NMR for monitoring subtle differences in binding. In contrast, the results with all the tested inhibitors manifest changes in the conformation of both the L3 and α3 regions. The PrOF NMR analyses reveal (for the first time) that it is possible to potently inhibit the MBLs by a mechanism not involving zinc ion chelation. The results are of interest for identifying novel inhibitor scaffolds for SPM-1, and by implication other MBLs, either for the design of non-β-lactam inhibitors that are not susceptible to β-lactamase hydrolysis and/or β-lactams/β-lactam analogues whose hydrolyzed products bind/inhibit MBLs. The overall results also clearly illustrate the utility of PrOF NMR for studying protein-ligand interactions in solution, detecting metal chelation (which is not always readily tractable in studies on metallo-enzyme), and revealing subtle differences in H-bonding and hydrophobic interactions.
Martine I Abboud
University of Oxford
Read the Original
This page is a summary of: 19
F-NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo-β-Lactamase, Angewandte Chemie International Edition, March 2017, Wiley,
DOI: 10.1002/anie.201612185.
You can read the full text:
Contributors
The following have contributed to this page
