A POINT MUTATION OF HUMAN NUCLEOSIDE DIPHOSPHATE KINASE-A FOUND IN AGGRESSIVE NEUROBLASTOMA AFFECTS PROTEIN-FOLDING

The point mutation serine 120 to glycine in the human nucleoside dipho sphate kinase A has been identified in several aggressive neuroblastom as (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 37 0, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymat ic and structural properties similar to the wild-type enzyme, protein lead whereas its stability to denaturation by heat and urea intermedia te. was markedly reduced. More importantly, upon renaturation of the u rea-denatured mutant protein, a folding intermediate accumulated, havi ng the characteristics of a molten globule. It had no tertiary structu re, as shown by near UV circular dichroism, whereas the secondary stru cture was substantially recovered. The hydrophobic probe 8-anilino-1-n aphthalene sulfonate bound to the intermediate species with an increas e in fluorescence intensity and a blue shift. The hydrodynamic size wa s between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturati on curve, and the protein showed the tendency to aggregate at the lowe st urea concentrations. The existence of a molten globule folding inte rmediates resulting from an altered folding in the mutated protein mig ht be related to the aggressiveness of neuroblastomas.