NMR Characterization of the Metallo-β-lactamase from Bacteroides fragilis and Its Interaction with a Tight-Binding Inhibitor:  Role of an Active-Site Loop

Abstract

Understanding the structure and dynamics of the enzymes that mediate antibiotic resistance of pathogenic bacteria will allow us to take steps to combat this increasingly serious public health hazard. Complete backbone NMR resonance assignments have been made for the broad-specificity metallo-beta-lactamase CcrA from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Chemical shift indices show that the secondary structure of the CcrA molecule in solution is very similar to that in published crystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears from the NMR experiments to be a regular beta-hairpin. Backbone heteronuclear NOE measurements indicate that this region has slightly greater flexibility on a picosecond to nanosecond time scale than the molecule as a whole. The loop appears to have an important role in the binding of substrates and inhibitors. Binding of the inhibitor 3-[2'-(S)-benzyl-3'-mercaptopropanoyl]-4-(S)-carboxy-5, 5-dimethylthiazolidine causes a marked increase in the stability of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-binding) site, including the beta-hairpin loop. There is a small but significant increase in the heteronuclear NOE for this loop upon inhibitor binding, indicative of a decrease in flexibility. In particular, the NOE of the indole ring of tryptophan 49, at the tip of the beta-hairpin loop, changes from a low value characteristic of a random coil chain to a significantly higher value, close to that observed for the backbone amides in this region of the protein. These results strongly suggest that the hairpin loop participates in the binding of substrate and in the shielding of the zinc sites from solvent. The broad specificity of the CcrA metallo-beta-lactamase may in fact reside in the plasticity of this part of the protein, which allows it to accommodate and bind tightly to substrates of a variety of shapes and sizes.

Crystal Structure of IMP-2 Metallo-β-lactamase from Acinetobacter spp.

“…Loop 1 is likely involved in the binding of substrates or inhibitors. 19,20) Loop 2, which connects a strand (β10) and a helix (α5), is composed of residues 224-240 (Fig. 2) and is located on approximately the opposite side of loop 1 centered around the Zn(II) ion-binding site.…”

“…9) NMR studies on CcrA by Scrofani et al suggest that Trp64 of IMP-1 plays a role in recruiting and stabilizing the substrate ligand. 19) The conformational flexibility of the GWG portion likely creates an open cavity in the active site, allowing the accommodation of a variety of bulky substrates. In contrast, judging from the 2|F o |−|F c | electron density map, the backbone of the GWG portion in IMP-2 is in a single conformation with a well-defined electron density (Fig.…”

Comparative Study of the Inhibition of Metallo-.BETA.-Lactamases (IMP-1 and VIM-2) by Thiol Compounds That Contain a Hydrophobic Group

“…A number of compounds have been examined as inhibitors of MBLs since 1996, including trifluoromethyl alcohols and ketones, 15) hydroxamates, 16) thiols, [17][18][19][20][21][22][23][24][25] thioester derivatives, 19,[26][27][28][29] cysteinyl peptides, 30) biphenyl tetrazoles, 31,32) mercaptocarboxylates, 12,21,29) 1b-methylcarbapenem derivatives, 33,34) 2,3-disubstituted succinic acid derivatives, 35) tricyclic natural products, 36) sulfonyl hydrazones, 37) and a synthetic cephamycin. 38) Most of these compounds, however, have been shown to exert their adverse effects only on a limited number of MBLs.…”

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

“…Considerable information exists regarding the B1 and B3 enzymes, including X-ray diffraction (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), spectroscopic (9,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), mechanistic (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59), and computational studies (60)(61)(62)(63)(64)…”