Allosteric Motions in Structures of Yeast NAD+-specific Isocitrate Dehydrogenase

Abstract

Mitochondrial NAD؉ -specific isocitrate dehydrogenases (IDHs) are key regulators of flux through biosynthetic and oxidative pathways in response to cellular energy levels. Here we present the first structures of a eukaryotic member of this enzyme family, the allosteric, hetero-octameric, NAD ؉ -specific IDH from yeast in three forms: 1) without ligands, 2) with bound analog citrate, and 3) with bound citrate ؉ AMP. The structures reveal the molecular basis for ligand binding to homologous but distinct regulatory and catalytic sites positioned at the interfaces between IDH1 and IDH2 subunits and define pathways of communication between heterodimers and heterotetramers in the hetero-octamer. Disulfide bonds observed at the heterotetrameric interfaces in the unliganded IDH hetero-octamer are reduced in the ligand-bound forms, suggesting a redox regulatory mechanism that may be analogous to the "on-off" regulation of non-allosteric bacterial IDHs via phosphorylation. The results strongly suggest that eukaryotic IDH enzymes are exquisitely tuned to ensure that allosteric activation occurs only when concentrations of isocitrate are elevated.The oxidative decarboxylation of isocitrate to ␣-ketoglutarate catalyzed by mitochondrial NAD ϩ -specific isocitrate dehydrogenases is a rate-limiting step in the tricarboxylic acid cycle. The affinities of the yeast and human enzymes (IDH, 5 EC 1.1.1.41) for isocitrate are allosterically regulated positively by AMP and ADP, respectively, and the human enzyme is also regulated negatively by ATP (1, 2). Both enzymes are negatively regulated by NADH (3, 4). This control has been proposed to contribute to inverse regulation of rates of energy production by oxidative pathways and by glycolysis (5) Yeast IDH is a hetero-octamer composed of four IDH1 (M r ϭ 38,001) and four IDH2 (M r ϭ 37,755) subunits with 42% sequence identity (6 -8) (supplemental Fig. 1). Results of targeted mutagenesis studies (9 -13) suggest that IDH2 contains a catalytic isocitrate/Mg 2ϩ -and NAD ϩ -binding site, whereas the homologous site in IDH1 functions in cooperative binding of isocitrate and in binding of the allosteric activator AMP. Yeast two-hybrid assays and other mutagenesis studies (13, 14) strongly suggest that a heterodimer of IDH1 and IDH2 is the fundamental structural building block of the enzyme and that IDH1 contributes a few residues to catalytic sites in IDH2, while the analogous residues of IDH2 function in the regulatory ligand-binding sites in IDH1. Visualization of subunit interactions within and between heterodimers in the holoenzyme is essential to define the molecular basis for allosteric communication between such sites and to provide information prerequisite for an understanding of the evolution of the highly regulated allosteric IDH hetero-octamer from the more ancient nonallosteric, homodimeric bacterial IDH enzymes. Mammalian NAD ϩ -specific isocitrate dehydrogenases are structurally more complex octamers than yeast IDH, containing three subunits in a ratio of ␣ 2 :␤:␥, with the ␣-s...

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