Monitoring conformational changes in the NDM-1 metallo-β-lactamase by 19F NMR spectroscopy

Abstract

The New Delhi metallo-β-lactamase (NDM-1) is involved in the emerging antibiotic resistance problem. Development of metallo-β-lactamases (MBLs) inhibitors has proven challenging, due to their conformational flexibility. Here we report site-selective labeling of NDM-1 with 1,1,1-trifluoro-3-bromo acetone (BFA), and its use to study binding events and conformational changes upon ligand-metal binding using (19) F NMR spectroscopy. The results demonstrate different modes of binding of known NDM-1 inhibitors, including L- and D-captopril by monitoring the changing chemical environment of the active-site loop of NDM-1. The method described will be applicable to other MBLs and more generally to monitoring ligand-induced conformational changes.

Keywords: 19F-NMR; NDM-1; antibiotic resistance; captopril; metallo-β-lactamases.

Figure 1

A) MBL-catalyzed β-lactam hydrolysis. B) Site-specific labeling of M67C NDM-1 with 3-bromo-1,1,1-trifluoroacetone (BFA) to give NDM-1*. ¹⁹F NMR spectra of NDM-1*-di-Zn^II complex, apo-NDM-1*, and denatured NDM-1* (obtained by incubation with 2 M guanidinium chloride) revealed distinctive signal pattern.

Figure 2

Monitoring of inhibitor binding to NDM-1 MBL by ¹⁹F NMR spectroscopy. A) Titration of NDM-1*-di-Zn^II (▴) with captopril stereoisomers leads to the appearance of distinctive peaks attributed to l- (▪) and D-captopril (•) complexes. l-Captopril can be displaced from a NDM-1* by D-captopril. B) The different chemical shifts for l- and D-captopril likely reflect different binding modes. View from crystallographic analyses of NDM-1 complexed with di-Zn^II and l-captopril (PDB id: 4EXS). The l-captopril (yellow) interacts with Asn220. Binding of D-captopril (pink) is modeled based on a structure of D-captopril complexed with the BlaB MBL (PDB id: 1M2X).

Figure 3

¹⁹F NMR spectroscopy enables identification of different modes of ligand binding. A) Titration of di-Zn^II-NDM-1* (70 μM) with thiols 1 and 2 (160 μM) implies different binding modes. B) Titration of di-Zn^II-NDM-1* (70 μM) with isoquinoline 3; averaged signal of di-Zn^II-NDM-1* non-bound and in complex with 3 indicates a fast exchange system.