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Chaperone action of a cofactor in protein folding.

ABSTRACT: 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.

SUBMITTER: Chen C

PROVIDER: S-EPMC7314391 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Visualizing chaperone-assisted protein folding.

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Project description:Chaperone saturation mediates translation and protein folding efficiency

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Project description:Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H(D). Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H(D). We find that K/J/E slows down RNase H(D)'s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H(D) with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.

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