A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding.

Ioan Lascu,Chanquing Wang,Manfred Konrad,Marie-Lise Lacombe,Claude Sarger,Gilberd Briand,Sabine Schaertl,Anna Giartosio

doi:10.1074/jbc.272.25.15599

Abstract

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.

Highlights

Nucleoside diphosphate (NDP)1 kinases, oligomeric enzymes made of small subunits, catalyze the reversible transfer of the terminal phosphate of nucleoside triphosphates to nucleoside diphosphates [1, 2]
We show here that altered folding properties of the recombinant S120G mutant protein lead to the accumulation of a molten globule intermediate
Full-length wild-type and S120G mutant NDP kinase A were expressed in bacteria in high yields and purified to apparent electrophoretic homogeneity

Summary

Introduction

Nucleoside diphosphate (NDP) kinases, oligomeric enzymes made of small subunits, catalyze the reversible transfer of the terminal phosphate of nucleoside triphosphates to nucleoside diphosphates [1, 2]. Data accumulated indicating that NDP kinases have regulatory functions. In Drosophila, NDP kinase, product of the awd gene, is essential for larvae growth and development [3, 4]. The decreased expression level of NDP kinase A, product of the Nm23-H1 gene has been correlated with the increased metastatic potential of some human tumors but not of others (for a review see Ref. 6). The other isoform, NDP kinase B, product of the Nm23-H2 gene, acts as a transcription factor of the oncogene c-myc, in vitro and in vivo [7,8,9]. The cDNA encoding for a forth human NDP kinase has recently been cloned [11].The last two proteins newly discovered are enzymatically active and possess amino-terminal extensions probably involved in their targeting to specific locations. We show here that altered folding properties of the recombinant S120G mutant protein lead to the accumulation of a molten globule intermediate

Methods

Results

Conclusion