NMR characterization of the metallo-beta-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor: Role of an active-site loop

Understanding the structure and dynamics of the enzymes that mediate antibi otic resistance of pathogenic bacteria will allow us to take steps to comba t this increasingly serious public health hazard. Complete backbone NMR res onance assignments have been made for the broad-specificity metallo-beta-la ctamase CcrA from Bacteroides fragilis in the presence and absence of a tig ht-binding inhibitor. Chemical shift indices show that the secondary struct ure of the CcrA molecule in solution is very similar to that in published c rystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears f rom the NMR experiments to be a regular beta-hairpin. Backbone heteronuclea r NOE measurements indicate that this region has slightly greater flexibili ty on a picosecond to nanosecond time scale than the molecule as a whole. T he 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 sta bility of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-bind ing) site, including the beta-hairpin loop. There is a small but significan t increase in the heteronuclear NOE for this loop upon inhibitor binding, i ndicative of a decrease in flexibility. In particular, the NOE of the indol e 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 th e protein. These results strongly suggest that the hairpin loop participate s in the binding of substrate and in the shielding of the zinc sites from s olvent. The broad specificity of the CcrA metallo-beta-lactamase may in fac t 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 .