Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization.

Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the wild type (WT) and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding and/or folding by hydrogen/deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are nativelike dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α0 ) remains unfolded. The Kpn/α0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.