Insights into the inhibitory mechanisms of NADH on the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase

Abstract

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the oxidative decarboxylation of isocitrate in the citric acid cycle. In the α₂βγ heterotetramer of NAD-IDH, the γ subunit plays the regulatory role and the β subunit the structural role. Previous biochemical data have shown that mammalian NAD-IDHs can be inhibited by NADH; however, the molecular mechanism is unclear. In this work, we show that the αβ, αγ and α₂βγ enzymes of human NAD-IDH can be inhibited by NADH, and further determine the crystal structure of the αγ heterodimer bound with an Mg²⁺ and an NADH at the active site and an NADH at the allosteric site, which resembles that of the inactive α^Mgγ heterodimer. The NADH at the active site occupies the binding site for NAD⁺ and prevents the binding of the cofactor. The NADH at the allosteric site occupies the binding sites for ADP and citrate and blocks the binding of the activators. The biochemical data confirm that the NADH binding competes with the binding of NAD⁺ and the binding of citrate and ADP, and the two effects together contribute to the NADH inhibition on the activity. These findings provide insights into the inhibitory mechanisms of the αγ heterodimer by NADH.

Figure 1

Inhibitory effects of NADH on the αγ, αβ and α₂βγ enzymes of human NAD-IDH. The enzymatic activities of the αγ and α₂βγ enzymes in the presence of NADH were measured at the standard conditions with varied concentrations of NADH as described in “Methods”. The activity of the αβ enzyme was determined at the same conditions except for a higher concentration of MnCl₂ (50 mM). The inhibitory effect of NADH on the activity of the enzyme is presented as percent of inhibition (I) as a function of NADH concentration. I = (V₀−V)/V₀ * 100, where V₀ and V are the initial velocities in the absence and presence of NADH, respectively.

Figure 2

Binding of NADH (a) and NAD⁺ (b) with the αγ, αβ and α₂βγ enzymes of human NAD-IDH measured by ITC at 20 °C. The concentrations of titrand (protein) and titrant (NADH or NAD⁺) in the measurements are listed in Table 1. The corrected heat change was obtained by subtracting the heat of dilution from the measured heat change, and the normalized corrected heat change of injectant was plotted against the molar ratio of titrant vs. titrand. The values of thermodynamic parameters were derived from the titration curve by fitting the experimental data using a nonlinear least-squares method with the single set of binding sites model and are summarized in Table 1. The dissociation constant K_d values are shown in the figure.

Figure 3

Structure of the α^Mg+NADHγ^NADH heterodimer of human NAD-IDH. (a) Overall structure of the α^Mg+NADHγ^NADH heterodimer in two different views. Left: view in perpendicular to the pseudo 2-fold axis of the αγ heterodimer. Right: view along the pseudo 2-fold axis of the αγ heterodimer. The color-coding schemes of individual domains of the α and γ subunits are shown above. The bound NADHs are shown in space-filling models, and the bound Mg²⁺ ion as a green sphere. (b) Representative simulated annealing composite omit maps (contoured at 1.0σ level) of the bound NADHs in the α^Mg+NADHγ^NADH structure. Left: Mg²⁺ and NADH at the active site. Right: NADH at the allosteric site. The NADH is shown in ball-and-stick model and the Mg²⁺ with a green sphere, respectively. (c) Structure-based sequence alignment of the α and γ subunits of human NAD-IDH with several representative NAD-IDHs. Homo sapien NAD-IDH: HsIDH3; S. cerevesiae NAD-IDH: ScIDH; Xenopus laevis NAD-IDH: XlIDH3; Danio rerio NAD-IDH: DrIDH3; Acidithiobacillus thiooxidans NAD-IDH: AtIDH. The secondary structures of the α and γ subunits in the α^Mg+NADHγ^NADH structure are placed on the top of the alignment. Invariant residues are highlighted by shaded red boxes and conserved residues by open blue boxes. The residues corresponding to those composing the ICT-binding site and the NAD-binding site in the αγ heterodimer of human NAD-IDH are highlighted with green and blue triangles, respectively.

Figure 4

Structures of the active site and the allosteric site in the α^Mg+NADHγ^NADH heterodimer. (a) Comparison of the ICT- and Mg²⁺-binding sites in the α^Mg+NADHγ^NADH (salmon), α^Mgγ (cyan, PDB code: 5GRH) and α^Mgγ^Mg+CIT+ADP (slate, PDB code: 5GRE) structures. The residues composing the active site are shown in stick models, and the metal ion and water molecules as spheres. For clarity, only the coordination bonds of the metal ion in the α^Mg+NADHγ^NADH structure are indicated with dashed lines. (b) Comparison of the NAD⁺/NADH-binding site at the active site in the α^Mg+NADHγ^NADH (salmon), α^Mgγ (cyan), α^Mgγ^Mg+CIT+ADP (slate), and A. thiooxidans NAD-IDH (green, PDB code: 2D4V) structures. For clarity, only the NADH in the α^Mg+NADHγ^NADH structure is shown in stick model, and its hydrogen-bonding interactions with the surrounding residues are indicated with dashed lines. (c) Structure of the allosteric site in the α^Mg+NADHγ^NADH structure. The NADH is shown in stick model, and its hydrogen-bonding interactions with the surrounding residues are indicated with dashed lines. (d) Comparison of the allosteric site in the α^Mg+NADHγ^NADH (left) and α^Mgγ^Mg+CIT+ADP (right) structures. The protein is shown with electrostatic potential surface, the bound NADH, CIT and ADP are shown with ball-and-stick models, and the Mg²⁺ with a green sphere. The NADH occupies a large portion of the binding sites for CIT, Mg²⁺ and ADP in the γ subunit. (e) Comparison of the allosteric site in the α^Mg+NADHγ^NADH (salmon), α^Mgγ (cyan), and α^Mgγ^Mg+CIT+ADP (slate) structures. The bound CIT in the α^Mgγ^Mg+CIT+ADP structure is shown in stick model and colored in gray, the metal ion is shown as a green sphere, and the hydrogen-bonding interactions between CIT and the surrounding residues are indicated with black dashed lines. The hydrogen-bonding interactions between Arg97^G-Tyr135^G and between Asn78^G-Arg272^G in the α^Mg+NADHγ^NADH structure are indicated with red dashed lines. (f) Comparison of the heterodimer interface in the α^Mg+NADHγ^NADH (salmon), α^Mgγ (cyan), and α^Mgγ^Mg+CIT+ADP (slate) structures. Left: the heterodimer interface in the α^Mg+NADHγ^NADH (salmon) and α^Mgγ (cyan) structures. Right: the heterodimer interface in the α^Mgγ^Mg+CIT+ADP (slate) structure. The hydrogen-bonding interactions between the N-terminal of the α7^A and α7^G helices and the β7^A and β7^G strands in the α^Mgγ^Mg+CIT+ADP structure are indicated with dashed lines.

Figure 5

Effects of NADH on the activity and the CIT or ADP activation of the αγ heterodimer. (a) The NAD⁺ saturation curve in the absence and presence of NADH. The kinetic parameters for NAD⁺ were measured at the standard conditions with varied concentrations of NAD⁺ (0–20 mM) as described in “Methods”. The V_max,NAD in the presence of 0, 50, and 100 μM NADH are determined to be 7.95 ± 0.13, 7.38 ± 0.20, and 6.79 ± 0.21 μmol/min/mg, respectively. The S_0.5,NAD values are shown in the figure. (b) The activation curve of CIT in the absence and presence of NADH. The CIT activation curves were measured at subsaturating substrate (ICT) conditions with varied concentrations of CIT as described in “Methods”. The V_max,CIT in the presence of 0, 50, and 100 μM NADH are determined to be 6.58 ± 0.14, 4.34 ± 0.11, and 2.63 ± 0.05 μmol/min/mg, respectively. The S_0.5,CIT values are shown in the figure. (c) The activation curve of ADP in the absence and presence of NADH. The ADP activation curves were measured at subsaturating substrate (ICT) conditions with varied concentrations of ADP in the presence of 1 mM CIT as described in “Methods”. The V_max,ADP in the presence of 0, 50, and 100 μM NADH are determined to be 14.1 ± 0.2, 13.0 ± 0.1, and 12.7 ± 0.2 μmol/min/mg, respectively. The S_0.5,ADP values are shown in the figure.