The point mutation
serine 120 to
glycine in the human
nucleoside diphosphate kinase A has been identified in several aggressive
neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant
nucleoside diphosphate kinase A. The mutant
enzyme had enzymatic and structural properties similar to the wild-type
enzyme, whereas its stability to denaturation by heat and
urea was markedly reduced. More importantly, upon renaturation of the
urea-denatured
mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse
urea gradient displayed no renaturation curve, and the
protein showed the tendency to aggregate at the lowest
urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated
protein might be related to the aggressiveness of
neuroblastomas.