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

• 成果类型: Article
• 署名作者: Williams, Christopher G.; Wang, Songlin; Carta, Veronica; Langan, Patricia S.; Thome, Alexander F.; Ramos, Sebastian A.; Holmes, Jacob B.; Ghosh, Rittik K.; Weiss, Kevin L.; Beran, Gregory J. O.; Hartman, Joshua D.; Coates, Leighton; Rienstra, Chad M.; Mueller, Leonard J.
• 署名单位: University of California System; University of California Riverside; University of Wisconsin System; University of Wisconsin Madison; University of Wisconsin System; University of Wisconsin Madison; United States Department of Energy (DOE); Oak Ridge National Laboratory
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2616547123
• 发表日期: 2026-09-22
• 页码: e2616547123
• 关键词: Toho-1 beta-lactamase; avibactam; NMR crystallography; MAS CryoProbe; UMA MLIP; CRYSTAL-STRUCTURE PREDICTION; CHEMICAL-SHIFT ANISOTROPY; CLASS-A; molecular-mechanism; amino-acids; x-ray; inhibition; h-1; intermediate; hydrolysis

摘要

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 beta-lactamase in complex with the non-beta-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 pKa perturbations, our data point to a more direct chemical origin arising from the intrinsic resistance of the Ser70-avibactam carbamoyl linkage to hydrolysis.