Chaperone action of a cofactor in protein folding.

Previously, we have reported that ATP accelerates the folding and unfolding of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a glycolytic enzyme utilizing NAD+ as a cofactor. Because ATP and NAD+ share the ADP moiety, we hypothesized that NAD+ also accelerates the folding of GAPDH and that the common structural motif between ATP and NAD+ is responsible for the chaperone activity. After confirming that NAD+ indeed accelerates the folding of GAPDH, we examined the chaperone activity of the structural fragments of NAD+ (ADP, AMP, adenosine, and nicotinamide monophosphate). Our finding showed that ADP and AMP significantly speed up the folding of GAPDH, while adenosine and nicotinamide monophosphate do not. ADP and AMP also dramatically speed up the unfolding of GAPDH by selectively stabilizing a transition state in which GAPDH has a partially unfolded conformation. Similar to the previously reported effect of ATP on the equilibrium unfolding of GAPDH, a partially unfolded intermediate also accumulates in the presence of ADP and AMP. Based on the effect of the structural fragments of NAD+ on the folding of GAPDH, we identified that AMP is the structural determinant of the chaperone activity of ATP and NAD+ . Also, we propose a plausible model to explain how NAD+ accelerates the folding of GAPDH through a stepwise development of molecular interactions with the protein.