Molecular mechanism of the allosteric regulation of the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase

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.

. Pair-wise superposition of the αγ heterodimer in different structures.
Overall RMSD (       The α and γ subunits in the α Mg γ structure are colored in lemon and cyan, and these in the α Mg γ Mg+CIT structure in magenta and orange, respectively. The IDH1 and IDH2 subunits in the apo structure (PDB code: 3BLX) are colored in pink and slate, and these in the CIT-bound structure (PDB code: 3BLV) in green and yellow, respectively. For easy comparison, we use the same secondary structure nomenclature for both human αγ heterodimer and yeast IDH1/IDH2 heterodimer. The structure elements and residues of the IDH1 and IDH2 subunits are superscripted by "1" and "2", respectively. The key residues are shown with side chains. The orientations of the α6 and α7 helices in both subunits are indicated with dashed arrows.

Figure S7. Structural comparison of the apo and CIT-bound IDH1/IDH2 heterodimer of yeast NAD-IDH.
(a) Comparison of the apo and CIT-bound IDH1/IDH2 structures at the allosteric site, the active site, and the heterodimer interface. The IDH1 and IDH2 subunits in the apo structure are colored in pink and slate, respectively; and these in the CIT-bound structure are colored in green and yellow, respectively. For easy comparison, we use the same secondary structure nomenclature for both human αγ heterodimer and yeast IDH1/IDH2 heterodimer. The structure elements and residues of the IDH1 and IDH2 subunits are superscripted by "1" and "2", respectively. The key residues are shown with side chains. Upon the binding of CIT, significant conformational changes are observed at the residues 78 1 -92 1 of IDH1, the C-terminal region of the β5-β6 loop and the β7 strand of both the IDH1 and IDH2 subunits at the heterodimer interface. The orientations of the α6 and α7 helices in both subunits are indicated with dashed arrows. The zoom-in panel on the right top shows the conformational changes of the β7 1 and β7 2 strands. For clarity, only the hydrogen-bonding interactions between the main chains of the residues are shown and the side chains are omitted.
(b) Structure of the allosteric site in the apo (left panel) and CIT-bound (right panel) IDH1/IDH2 structures. In the apo structure, residues 78-92 adopt a helical conformation which block the CITbinding site; in the CIT-bound structure, residues 78-92 adopt a loop conformation. The hydrophilic interactions between CIT and the surrounding residues are indicated with dashed lines and several key residues are shown with side chains.
(c) Structure of the heterodimer interface in the apo (left panel) and CIT-bound (right panel) IDH1/IDH2 structures. The structure elements at the heterodimer interface that undergo major conformational changes upon the CIT binding include the β5-β6 loop and the β7 strand of both IDH1 and IDH2 subunits. Several key residues are shown with side chains. For clarity, only the hydrogen-bonding interactions that are altered upon the CIT binding are indicated with dashed lines. (d) A schematic diagram showing the hydrogen-bonding interactions among the β5-β6 loop, the α7 helix, the β7 strand, and the α5 helix of both IDH1 and IDH2 subunits in the apo and CIT-bound IDH1/IDH2 structures. The interactions in the apo structure are indicated with green lines; these disrupted upon the CIT binding are indicated with dashed green lines, and the newly formed interactions are indicated with red lines.