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/c4sc01752hClick here for additional data file

Abstract

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, however, reveal that SPM-1 possesses loop and mobile element regions characteristic of the B2 MBLs. These include a mobile α3 region which is important in catalysis and determining inhibitor selectivity. SPM-1 thus appears to be a hybrid B1/B2 MBL. The results have implications for MBL evolution and inhibitor design.

Fig. 1. (A) Outline mode of action of metallo-β-lactamases (MBLs); (B–E) views from MBL crystal structures highlighting potentially mobile regions. Views of (B) IMP-1 (B1 MBL, PDB ID: 1JJT), (C) CphA (B2 MBL, PDB ID: ; 1X8I) and the (D) ‘open’ and (E) ‘closed’ forms of SPM-1 (B1 MBL, ‘open’ PDB ID: ; 2FHX and ‘closed’ PDB ID: ; 4BP0) highlighting the different mobile regions (L3 loop (blue), L10 loop (red) and α3 region (green)) that characterize the MBL subfamilies. A longer L3 loop (blue) is characteristic of the di-Zn B1 MBLs. The B2 MBLs using mono-zinc ion are characterised by an elongated α3 region (green) and a shorter L3 loop (blue). In the case of the B1 MBLs only SPM-1 has an elongated α3 loop (green) and a short L3 loop (blue).

Fig. 2. Crystallographic evidence for conformational changes in SPM-1. (A) Comparison of SPM-1 structures with the α3 region in an ‘open’ (PDB ID: 2FHX) and ‘closed’ (PDB ID: ; 4BP0) conformation. (B) Representative 2Fo–Fc electron density contoured to 1σ (blue mesh) of the active site residues and Zn atoms for the ‘closed’ SPM-1 structure. (C) Superimposition of CphA (purple, PDB ID: ; 1X8I) and the ‘closed’ SPM-1 structure (green), showing the position of hydrolysed biapenem in the active site.

Fig. 3. Non-denaturing ion mobility MS reveals different conformations of di-Zn(ii)-SPM-1 as compared to the theoretical collisional cross section (CCS) values of the ‘open’ and ‘closed’ crystal forms.

Fig. 4. The loop of SPM-1 adopts distinct conformations in solution. (A) ¹⁹F-NMR spectra of folded di-Zn(ii)-SPM-1* (top) and denatured SPM-1* (bottom) revealed distinct signals (The peak labeled * may correspond to an intermediate state between non-denatured and denatured states); (B) views of the SPM-1 α3 region in ‘closed’ and ‘open’ conformations, showing the different position of Tyr-152 (the residue which was substituted for Cys, derivatized with CF₃COCH₂Br, to give a Enz–SCH₂COCF₃ species).

Fig. 5. 1D selective magnetization transfer experiments recorded at 310 K enable the exchange rate of loop movement to be determined. (A) 1D ¹⁹F exchange NMR spectra. (B) Fitted exchange data for SPM-1* (peak A = –82.2 ppm, peak B = –71.4 ppm). The solid curves are derived from the CIFIT fits.

Fig. 6. Stopped-flow analysis of SPM-1 catalysed meropenem hydrolysis. (A) Apo-SPM-1 (100 μM) was incubated with Zn(ii) (200 μM) for ∼10 min then mixed with meropenem (100 μM) (5 °C); (B) apo-SPM-1 (100 μM) was incubated with Co(ii) (500 μM) for ∼10 min, then mixed with meropenem (100 μM); (C) global fit traces and proposed kinetic mechanism. Global fit traces and proposed kinetic mechanism (assuming that only di-Co(ii) substituted SPM-1 (E) is present in solution before the reaction due to 5-fold excess of Co(ii) used for the experiment).