The Killer-of-prune (K-pn) mutation in Drosophila corresponds to a Pro-Ser substitution in nucleoside diphosphate kinase (Lascu, I. Chaffotte, A., Limbourg-Bouchon, B., and Véron, M. (1992) J. Biol. Chem. 267, 12775-12781). We investigated the role of the equivalent proline (Pro100) in the formation and stability of the Dictyostelium nucleoside diphosphate kinase hexamers. Mutations to serine or glycine had only little effect on the properties of the native enzyme. However, the mutant drastically affected the subunit interaction in the hexamer and the ability of the isolated subunits to associate in vitro. While the wild-type hexamer inactivated and unfolded concomitantly at 5-6 M urea, the mutant proteins dissociated to monomers at 0.5-2 M urea and unfolded at 2.5-4 M urea. At intermediate urea concentrations, the unique species present in solution was a folded, partially active monomer as shown by size-exclusion chromatography, UV, fluorescence, and CD spectroscopy. Proline 100 is located in a loop involved in subunits contact. Altered conformation of the loop in P100S and P100S mutants demonstrates its crucial role in subunit assembly. We propose to explain the conditional dominance of the K-pn mutation by the presence of a monomeric form of the enzyme that would have deleterious effects in vivo.