Molecular basis for the function of the ?? heterodimer of human NAD-dependent isocitrate dehydrogenase

Mammalian mitochondrial NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the decarboxylation of isocitrate into ?-ketoglutarate in the tricarboxylic acid cycle. It exists as the ?(2)?? heterotetramer composed of the ?? and ?? heterodimers. Different from the ?? heterodimer that can be allosterically activated by CIT and ADP, the ?? heterodimer cannot be allosterically regulated by the activators; however, the molecular mechanism is unclear. We report here the crystal structures of the ?? heterodimer of human NAD-IDH with the ? subunit in apo form and in Ca2+-bound, NAD-bound, and NADH-bound forms. Structural analyses and comparisons reveal that the ?? heterodimer has a similar yet more compact overall structure compared with the ?? heterodimer and contains a pseudo-allosteric site that is structurally different from the allosteric site. In particular, the ?3-?3 and ?12-?8 loops of the ? subunit at the pseudo-allosteric site adopt significantly different conformations from those of the ? subunit at the allosteric site and hence impede the binding of the activators, explaining why the ?? heterodimer cannot be allosterically regulated by the activators. The structural data also show that NADH can compete with NAD to bind to the active site and inhibits the activity of the ?? heterodimer. These findings together with the biochemical data reveal the molecular basis for the function of the ?? heterodimer of human NAD-IDH.