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 nuclear magnetic resonance (NMR) spectroscopy. 15N relaxation experiments show that BlaC is mostly rigid on the pico- to nanosecond timescale. Saturation transfer experiments indicate that also on the high-millisecond timescale 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 (exchange rate, kex, of <100 s−1), while for this inhibitor also loss of pico- to nanosecond timescale 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.

Mechanisms of ligand binding

“… 132 Systems that have been assigned to IF include the Mycobacterium tuberculosis β-lactamase that is mostly rigid in the free form, as established by 15 N relaxation experiments, but becomes more dynamic upon binding of the antibiotic avibactam. 140 IF has been invoked as a mechanism to optimize molecular switches such as aptamers that change their conformation upon target binding to benefit applications in biotechnology and synthetic biology, 141 as well as a mechanism for the Asp symporter opening upon Na + binding, 142 for an archaeal homolog of the excitatory amino acid transporter involved in glutamatergic synaptic transmission in the mammalian central nervous system 143 and for selective inhibitors of the FK506-binding protein 51. 144 A number of systems, on the other hand, are more consistent with CS.…”

Two β-Lactamase Variants with Reduced Clavulanic Acid Inhibition Display Different Millisecond Dynamics

“…We previously reported no measurable exchange in this regime for wt BlaC. 10 CEST of the G132N mutant was measured, yielding direct observation of exchange between the resonances with the largest chemical shift differences, Gly238 and Asp246 (Figure S10).…”

“…The phenomenon of split resonance peaks in the G132N spectra is reminiscent of that in the clavulanic acid adduct-bound wt protein. 10 However, the relative positions of the peaks are not similar, nor could any exchange be determined between the adduct-bound states. Moreover, the number of observed states for the adduct-bound protein is not the same as that for the G132N mutant.…”

“…We recently reported dynamics in the active site of BlaC, both in the resting state and when bound to clavulanic acid or avibactam adducts. 10 We wondered whether the dynamic behavior was affected by the G132N and K234R mutations. Therefore, the 1 H- 15 N correlation spectra of BlaC mutants G132N and K234R were assigned using HNCa spectra in combination with the assignments for wt BlaC, for which peaks have been assigned to all non-proline backbone amides except Asp2 and four residues in the active site, close to the phosphate binding site.…”

“…We have recently characterized the dynamic behavior of the β-lactamase of Mycobacterium tuberculosis , BlaC ( Fig. 1 ), and shown that this enzyme indeed features pronounced millisecond dynamics around its active site ( 10 ). It can be hypothesized that apart from allowing the enzyme to hydrolyze its extraordinarily broad spectrum of substrates, this flexibility may aid the enzyme to evolve more readily to gain new functionalities.…”