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bioRxiv · 10.1101/2025.10.09.681369

Quaternary structures of Streptococcus pneumoniae Nucleoside Diphosphate Kinase: from hexamers to supramolecular assemblies

KERBOEUF, J.·NOURI, P.·GALISSON, F.·DAURY, L.·GIRAUD, M.-F.·LAMBERT, O.·ORELLE, C.·BALLUT, L.·KAPLAN, E.·JAULT, J.-M.·GONZALEZ, C.
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Abstract

The Nucleoside Diphosphate Kinase (NDK) is a key enzyme that controls the balance of nucleotide pools in all living organisms. Beyond this fundamental role, NDKs exert pleiotropic effects in many cellular processes, including cell development, signal transduction, differentiation, tumor metastasis, and gene expression. The quaternary structure of NDK is typically hexameric--organized as a trimer of dimers in eukaryotic cells and in many prokaryotes--though in some species it can also be found as a tetramer (a dimer of dimers). Here, we report the crystal structure of Streptococcus pneumoniae NDK (SpNDK) in its apo state (1.2 [A]) and in an ADP-vanadate-bound state (3.4 [A]). In both structures, SpNDK adopts a hexameric assembly and the fold of each monomer is highly conserved compared to NDKs from other organisms. A notable feature is the extended Kpn-loop, which plays a key role in stabilizing the hexamer. Interestingly, the thermal stability of SpNDK was remarkably high (~75 {degrees}C), a surprising feature given the mesophilic lifestyle of S. pneumoniae. Unexpectedly, size-exclusion chromatography revealed that SpNDK exists as an equilibrium mixture of hexamers, dodecamers, and higher-order supramolecular assemblies, and this was further confirmed by mass photometry. Using cryo-electron microscopy, we solved the 3D structure of the hexameric state at 2.47 [A] resolution, and resolved dodecameric assemblies of the protein. Since the oligomeric state of the NDK influences its cellular function, our findings suggest that supramolecular assemblies could modulate the activity of SpNDK in vivo, aligning with the broad and multifaceted roles of this enzyme family.

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KERBOEUF, J., NOURI, P., GALISSON, F., DAURY, L., GIRAUD, M.-F., LAMBERT, O., ORELLE, C., BALLUT, L., KAPLAN, E., JAULT, J.-M., GONZALEZ, C.. 2025-10-09. Quaternary structures of Streptococcus pneumoniae Nucleoside Diphosphate Kinase: from hexamers to supramolecular assemblies. https://doi.org/10.1101/2025.10.09.681369

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