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 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.
Lascu
I
I
Université de Bordeaux-2, Institut de Biochimie et Génétique Cellulaires-CNRS, 33077 Bordeaux, France. ioan.lascu@ibgc.u-bordeaux2.fr
Schaertl
S
S
Wang
C
C
Sarger
C
C
Giartosio
A
A
Briand
G
G
Lacombe
M L
ML
Konrad
M
M
D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005998:Glycine; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D020559:Monomeric GTP-Binding Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D009447:Neuroblastoma / Q000201:enzymology* / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics*; D017354:Point Mutation; D017510:Protein Folding; D012694:Serine; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
D000951:Antigens, Neoplasm; D054778:NM23 Nucleoside Diphosphate Kinases; D014157:Transcription Factors; D012694:Serine; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins; D005998:Glycine