Modelling dynamics of human NDPK hexamer structure, stability and interactions

Nucleoside diphosphate kinases (NDPKs) are evolutionarily conserved multifunctional enzymes involved in energy metabolism and gene regulation. NDPKs primarily regulate nucleotide pool turnover by catalyzing the transfer between nucleoside triphosphate (NTPs) and their deoxy derivatives, maintaining cellular homeostasis. The NDPK hexameric assembly is needed for kinase activity, but its precise assembly into homo-/hetero-oligomeric complexes remains poorly understood. How quaternary structure affects NDPK activity is limited by high subunit homology, experimental challenges in isolating in vivo heterohexamers and subunit abundances across cellular compartments. We identify conserved Arg27 across group I NDPKs (NME1-4) as the key residue for hexamer assembly. The Arg27 ensures similar hexameric assembly across subunits and mediates inter- and intra-molecular monomeric interactions, while Arg27 mutation leads to decreased binding affinity, dynamics, and complex destabilization. The double and triple Arg NME4 mutations destabilize hexamer into dimer due to shorter C-terminal region. Simulating NME1-3 with Arg mutations and shortened C-terminal recapitulates hexameric destabilization, highlighting role of the C-terminal region in stabilizing NDPK hexamers. Comparing heterohexameric complexes, we report NME1-NME2 (A ₁ B ₅ ) complex as most stable and abundant, owing to predominant subunit nuclear localization. We propose that Arginine residues, C-terminal sequence and subunit abundances contribute to formation and stabilization of NDPK heterohexameric complexes.