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. 2021 Jun 25;297(2):100918. doi: 10.1016/j.jbc.2021.100918

Figure 4.

Figure 4

Maleimide probes detect mononuclear MBLs through the action of AMA both in vitro and in live bacteria.A, structure of mono-Zn2+ NDM-1 illustrating the Zn2+ binding sites (Protein Data Bank ID: 3SFP). The Zn1 and Zn2 sites are defined by H122, H120, and H189 and D124, C208, and H250, respectively. B, representative in vitro time course of NDM-1 (1 μM) dansylation in assay buffer (25 mM Hepes–NaOH, 1% [v/v] PEG 4000, 10 μM ZnSO4, and pH 7.5) with AMA (0.1 mM) at 37 °C. At the end of the time course, ZnSO4 (0.11 mM) or ZnSO4 and SDS (2% w/v) were added to separate aliquots of the reaction. Top, SDS-PAGE analysis with fluorescence detection (FL) shows the increasing labeling of NDM-1 (1 μM) in the presence of DM (0.5 mM) and AMA (0.1 mM). NDM-1 is not labeled in the presence of ZnSO4 and entirely labeled when denatured with SDS. Bottom, Coomassie brilliant blue (CBB) staining of total protein. C, quantitative analysis of maleimide labeling (blue) in panel (B) relative to enzyme activity loss under the same conditions without DM present (cyan). The data are representative of two replicates, and the error bars represent SD. D, intact protein LC ESI–MS analyses of NDM-1 (1 μM) treated with B7–Mal following incubation (1 h) in buffer either lacking (black trace) or containing (red trace) AMA (0.5 mM). E, schematic diagram of a maleimide-based method to enable detection of mononuclear MBLs. AMA causes protein-bound Zn2+ to dissociate exposing the side chain of Cys208 that is involved in metal coordination in class B1 MBLs. A maleimide probe then modifies the Cys208 allowing for detection through reporter molecules (R = dansyl or biotin). F, in situ BM labeling and immunoprecipitation of NDM-1FLAG. Escherichia coli–producing NDM-1FLAG was treated (15 min) with (+) or without (−) AMA (25 μM) and l-captopril (0.15 mM) for 15 min. Following this, cells were incubated with BM (0.5 mM; 15 min). Biotinylated protein and total NDM-1 were detected with streptavidin–HRP (strep) and anti-FLAG-HRP IgG (αFLAG), respectively. Panel F is representative of two independent experiments. AMA, aspergillomarasmine A; B7–Mal, biotin–maleimide; DM, N-[2-(dansylamino)ethyl]maleimide; ESI–MS, electrospray ionization–MS; FL, fluorescence; HRP, horseradish peroxidase; MBL, metallo-β-lactamase; NDM-1, New Delhi MBL-1.