Molecular mechanism of the functions and allosteric regulations of the αβ and αγ heterodimers of human NAD‐dependent isocitrate dehydrogenase
Authors: Ding Jianping , Ma Tengfei , Liu Yabing , Peng Yijie , Huang Wei 
Published: November 2023

Human NAD‐dependent isocitrate dehydrogenase, existing as the α2βγ heterotetramer, catalyzes the decarboxylation of isocitrate into α‐ketoglutarate in the Krebs cycle, which can be allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the individual subunits and heterodimers, we systematically characterized the enzymatic properties of the α2βγ heterotetramer and αβ and αγ heterodimers of NAD‐IDH and performed the structural and functional studies of the αβ and αγ heterodimers. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of α2βγ requires the assembly and cooperative function of both heterodimers. α2βγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S0.5,ICT and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Furthermore, the allosteric activation of α2βγ is through the γ subunit not the β subunit. These results indicate that the γ subunit plays the regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme.

Structural studies of the αγ heterodimer show that CIT, ADP and Mg2+ bind adjacent to each other at the allosteric site in the γ subunit. 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. Structural studies of the αβ heterodimer show that the β subunit contains a pseudo allosteric site different from the γ subunit, explaining its incapability to bind activators. However, the αβ heterodimer exhibits similar heterodimer and heterotetramer interfaces as the αγ heterodimer. Mutagenesis and kinetic data show that key residues at the allosteric site and the interfaces play important roles in the activation of the holoenzyme but these at the pseudo allosteric site do not. The structural and biochemical data together indicate that there is a structural communication between the allosteric site of the γ subunit and the active sites of both heterodimers via the interfaces, and the binding of activators to the allosteric site can regulate both α subunits. These findings reveal the molecular mechanism of the functional roles and the allosteric regulations of the αγ and αβ heterodimers of human NAD‐IDH.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (31170690 and 31521061), the Ministry of Science and Technology of China (2013CB910404) and the Chinese Academy of Sciences (XDB08010302).