Spotlight on the Nucleotide: Solid-State NMR for the Investigation of ATP Hydrolysis in the ATPase SmsC

Solid-state nuclear magnetic resonance (NMR) spectroscopy is an ideal tool to study ATP hydrolysis in ATP-driven processes at atomic resolution. In this study, we present a nucleotide-detected solid-state NMR approach to probe nucleotide conformations and dynamics during ATP hydrolysis using the dimeric P-loop ATPase SmsC as a model protein. ³¹P-detected solid-state NMR experiments under magic-angle spinning (MAS) conditions provide insights into the different stages of ATP hydrolysis, which have been mimicked by ATP analogues that are often considered as "nonhydrolyzable". Our data reveal that different degrees of conformational heterogeneity are observed for such ATP mimics. By computational modeling, we explore the role of a hydrogen bond between SmsC and the terminal phosphate group of the nucleotides in defining the nucleotide binding conformation. Homonuclear ³¹P-³¹P dipolar coupling constant measurements for the bound ATP analogues have been performed to obtain information about their dynamic properties. These data show that the ATP and the transition-state analogues maintain some molecular motion, while the post-hydrolytic mimic AMPCP shows significantly less dynamics. The approach presented herein for investigating ATP hydrolysis can be easily transferred to other ATP-fueled proteins, including difficult to express proteins or large protein complexes, therefore providing a way to functionally characterize these systems.