Molecular mechanism of the dual regulatory roles of ATP on the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Pengkai Sun,Jianping Ding,Tuya Bai,Tengfei Ma

doi:10.1038/s41598-020-63425-6

Abstract

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.

Highlights

The Krebs cycle is a central part of the metabolic pathway to generate ATP through the oxidation of acetyl-CoA derived from the chemical breakdown of carbohydrates, fats and proteins
We determined two crystal structures of the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase (NAD-Isocitrate dehydrogenases (IDHs)) with the active site bound with either Mg2+ alone or Mg2+ and ATP, and the allosteric site bound with Mg2+, CIT and ATP
Soaking the crystals of the αMgγMg+CIT heterodimer in a solution containing a very high concentration of ATP (200 mM) yielded crystals of the αγ heterodimer with the active site bound with Mg2+ and ATP and the allosteric site bound with Mg2+, CIT and ATP, which led to the determination of the structure of the αMg+ATPγMg+CIT+ATP heterodimer

Summary

Introduction

The Krebs cycle is a central part of the metabolic pathway to generate ATP through the oxidation of acetyl-CoA derived from the chemical breakdown of carbohydrates, fats and proteins. The activities of mammalian NAD-IDHs can be regulated by many metabolic intermediates and products of the Krebs cycle via feedback activation and inhibition mechanisms. In the αMgγMg+CIT+ATP structure, there is ATP bound to the ADP binding site of the allosteric site, which stabilizes CIT binding through Mg2+-mediated interactions and leads the enzyme to adopt an active conformation. Our biochemical data show that high concentrations of ATP facilitate the formation of metal ion-ATP chelates and decrease the concentration of free metal ion available for the catalytic reaction, inhibiting the enzymatic activity. Our structural and biochemical data together reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH

Methods

Results

Conclusion