Toho-1 β-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam

Backbone chemical shift assignments for the Toho-1 beta-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly H-2,C-13,N-15-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-beta-lactam beta-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of N-15 relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of similar to 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

Automated summary

The backbone chemical shift assignments of Toho-1 beta-lactamase were determined using NMR experiments, allowing for site-specific characterization. The study revealed high rigidity on short timescales and active site flexibility on longer timescales, which is consistent with achieving high catalytic efficiency and broad substrate specificity. These findings suggest that induced active site dynamics in the enzyme play a crucial role in accommodating variously sized substrates and sampling the optimal conformation for catalysis.