β-Lactamase of Mycobacterium tuberculosis shows dynamics in the active site that increase upon inhibitor binding

Abstract

The Mycobacterium tuberculosis β-lactamase BlaC is a broad-spectrum β-lactamase that can convert a range of β-lactam antibiotics. Enzymes with low specificity are expected to exhibit active site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using NMR spectroscopy. 15N relaxation experiments show that BlaC is mostly rigid on the pico- to nanosecond time scale. Saturation transfer experiments indicate that also on the high millisecond time scale BlaC is not dynamic. Using relaxation dispersion experiments, clear evidence was obtained for dynamics in the low millisecond range, with an exchange rate of ca. 860 s-1 The dynamic amide groups are localized in the active site. Upon formation of an adduct with the inhibitor avibactam, extensive line broadening occurs, indicating an increase in magnitude of the active site dynamics. Furthermore, the rate of the motions increases significantly. Upon reaction with the inhibitor clavulanic acid, similar line broadening is accompanied by duplication of NMR signals, indicative of at least one additional, slower exchange process (kex < 100 s-1), while for this inhibitor also loss of pico- to nanosecond time scale rigidity is observed for some amides in the α-domain. Possible sources of the observed dynamics, such as motions in the omega loop and rearrangements of active site residues, are discussed. The increase in dynamics upon ligand binding argues against a model of inhibitor binding through conformational selection. Rather, the induced dynamics may serve to maximize the likelihood of sampling the optimal conformation for hydrolysis of the bound ligand.