NMR crystallography reveals active-site protonation states of Toho-1 β-lactamase in complex with avibactam

The determination of active-site protonation states is critical for a full mechanistic understanding of enzyme catalysis and inhibition. Here, we employ NMR crystallography-the integrated combination of solid-state NMR spectroscopy, X-ray diffraction, and first-principles computational chemistry-to determine the protonation states of the active site of Toho-1 β-lactamase in complex with the non-β-lactam inhibitor avibactam. We report two X-ray crystal structures of the Toho-1:avibactam complex, along with high-field solid-state NMR measurements that enable near-complete backbone and side-chain resonance assignments. To overcome the computational scaling limits that have traditionally hindered NMR crystallography in large systems, we use an accelerated workflow in which machine-learning interatomic potentials enable efficient geometry refinement prior to density functional theory chemical shift calculations. For Toho-1, quantitative analysis of the active-site chemical shifts and chemical shift tensors using this hybrid protocol reveals that the key active-site side chains retain their canonical charge states in the presence of avibactam, with Lys73 and Lys234 protonated and positively charged, and Glu166 deprotonated and poised to function as a general base. Contrary to recent proposals suggesting that avibactam inhibits by suppressing essential proton transfers through pK_a perturbations, our data point to a more direct chemical origin arising from the intrinsic resistance of the Ser70-avibactam carbamoyl linkage to hydrolysis.