Molecular mechanism of the allosteric regulation of the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Tengfei Ma,Yingjie Peng,Jianping Ding,Wei Huang

doi:10.1038/srep40921

Abstract

Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Previously, we have demonstrated biochemically that the α2βγ heterotetramer and αγ heterodimer can be allosterically activated by citrate (CIT) and ADP. In this work, we report the crystal structures of the αγ heterodimer with the γ subunit bound without or with different activators. Structural analyses show that CIT, ADP and Mg2+ bind adjacent to each other at the allosteric site. The CIT binding induces conformational changes at the allosteric site, which are transmitted to the active site through the heterodimer interface, leading to stabilization of the ICT binding at the active site and thus activation of the enzyme. The ADP binding induces no further conformational changes but enhances the CIT binding through Mg2+-mediated interactions, yielding a synergistic activation effect. ICT can also bind to the CIT-binding subsite, which induces similar conformational changes but exhibits a weaker activation effect. The functional roles of the key residues are verified by mutagenesis, kinetic and structural studies. Our structural and functional data together reveal the molecular mechanism of the allosteric regulation of the αγ heterodimer.

Highlights

Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle
We found that the CIT binding induces significant conformational changes at the allosteric site, which are transmitted to the active site through the heterodimer interface, leading to stabilization of the ICT binding at the active site and activation of the enzyme
The β7G strand has no conformational change, and the β7A strand, the β5A–β6A loop and the N-terminal region of the α7A helix have no conformational changes and adopt similar conformations as in the αMgγstructure (Fig. 4a,c and Supplementary Figure S4a,c). These results demonstrate that the αγK 151A mutant can bind CIT and ADP and induce conformational changes of the allosteric site and some structural elements of the heterodimer interface in the γsubunit, the γ-K151A mutation disrupts the structural communication between the γsubunit and the αsubunit and the conformational changes at the allosteric site cannot be transmitted to the active site

Summary

Introduction

Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Isocitrate dehydrogenases (IDHs) are a family of enzymes that catalyze the oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (α-KG) using NADP or NAD as coenzyme. Most bacteria and archaea contain only NADP-dependent IDHs (NADP-IDHs, EC 1.1.1.42) in the cytosol, which carry out the catalytic reaction in the Krebs or tricarboxylic acid (TCA) cycle. The molecular mechanisms of the function and catalytic reaction of NADP-IDHs have been extensively studied at the biochemical and structural levels. The crystal structures of NADP-IDHs from various species, including E. coli NADP-IDH (EcIDH), porcine mitochondrial NADP-IDH (PmIDH) and human cytosolic NADP-IDH www.nature.com/scientificreports/

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