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

Abstract

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 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/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 native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 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.

Structure, Folding and Stability of Nucleoside Diphosphate Kinases

“…The C-terminal part (that is, the residues extending after the α 4 helix) is one of the most mobile and variable areas of NDPK and is involved in the dimerization and trimerization of the majority of hexamers. Its length varies from 18 residues in human isoforms to 4 residues in Mycobacterium tuberculosis ( M. tuberculosis ) [ 36 , 37 ]. In contrast, the C-terminus is shorter in tetramers (see alignment, Figure 1 ) and is not involved in the quaternary structure.…”

“… Structure of monomer, dimer, trimer, and hexamer of NDPK from M. tuberculosis (Figure adapted with permission from [ 36 ]). ( A ) View of the monomer with labeled secondary structural elements.…”

“…We recently confirmed this hypothesis using HDX-MS technology at the peptide level. Characterizing stable dimers from M. tuberculosis , we showed that they were native-like, except for a part of the binding site we called Kpn /α 0 subdomain which remained unfolded [ 36 ]. These dimers were unable to get phosphorylated presumably due to the fact that their binding sites are not fully constituted.…”

“…For these studies, the folded and the unfolded NDPK was incubated for 12 to 24 h in the presence of different amounts of denaturant to induce, respectively, the unfolding and the refolding of NDPK and enzyme activity or intrinsic fluorescence followed. Many of these studies compared the stability of the WT versus different mutants and concluded that: (1) most mutations had a destabilizing effect [49,59,61,62,69] and (2) the quaternary structure of thermophilic or hyper-thermophilic NDPK is stabilized by interactions between monomer subunits [36,41,46]. Quantifying thermodynamically the stabilization or the destabilization effect was not possible in most of these studies since the NDPK was not reaching equilibrium during unfolding/refolding but experienced a hysteresis phenomenon [66] and no thermodynamic parameter (∆G, ∆H or ∆S) could be extracted.…”

Remodeling of the Binding Site of Nucleoside Diphosphate Kinase Revealed by X-ray Structure and H/D Exchange

“…When the bridges were abolished by the D93N point mutation, the mutant was still hexameric and enzymatically active, but its T m dropped by 28 °C . Equilibrium analysis of the wild type (WT) and D93N mutant by hydrogen/deuterium exchange mass spectrometry (HDX-MS) allowed us to show that the R80–D93 bridge was not only important with respect to hexamer stability but also influenced its conformational dynamics …”