Extrinsic conditions influence the self-association and structure of IF1, the regulatory protein of mitochondrial ATP synthase

Abstract

The endogenous inhibitor of ATP synthase in mitochondria, called IF₁, conserves cellular energy when the proton-motive force collapses by inhibiting ATP hydrolysis. Around neutrality, the 84-amino-acid bovine IF₁ is thought to self-assemble into active dimers and, under alkaline conditions, into inactive tetramers and higher oligomers. Dimerization is mediated by formation of an antiparallel α-helical coiled-coil involving residues 44-84. The inhibitory region of each monomer from residues 1-46 is largely α-helical in crystals, but disordered in solution. The formation of the inhibited enzyme complex requires the hydrolysis of two ATP molecules, and in the complex the disordered region from residues 8-13 is extended and is followed by an α-helix from residues 14-18 and a longer α-helix from residue 21, which continues unbroken into the coiled-coil region. From residues 21-46, the long α-helix binds to other α-helices in the C-terminal region of predominantly one of the β-subunits in the most closed of the three catalytic interfaces. The definition of the factors that influence the self-association of IF₁ is a key to understanding the regulation of its inhibitory properties. Therefore, we investigated the influence of pH and salt-types on the self-association of bovine IF₁ and the folding of its unfolded region. We identified the equilibrium between dimers and tetramers as a potential central factor in the in vivo modulation of the inhibitory activity and suggest that the intrinsically disordered region makes its inhibitory potency exquisitely sensitive and responsive to physiological changes that influence the capability of mitochondria to make ATP.

Keywords: ATP hydrolysis; inhibitor; intrinsically disordered protein; mitochondria; regulation.

Fig. 1.

Changes in secondary structure induced by pH. (A and B) CD spectra of IF₁-Y33W and IF₁-Y33W-H49K, respectively, at protein concentrations of 10 μM and pH values of 2.4 (gray), 4.0 (orange), 4.6, (green) and 6.0 (blue). (C and D) Dependence of helicity of IF₁-Y33W and IF₁-Y33W-H49K, respectively, on pH at various protein concentrations. IF₁-Y33W: 1 μM (light blue line); 10 μM (medium blue line); 50 μM (light purple line); 100 μM (dark blue line). IF₁-Y33W-H49K: 1 μM (pink line); 10 μM (red line); 50 μM (brown line); 100 μM (maroon line). In C and D, error bars denote the range of values measured.

Fig. 2.

Chemical denaturation of IF₁-Y33W and IF₁-Y33W-H49K. (A and B) Urea denaturation at pH 7.0 of IF₁-Y33W and IF₁-Y33W-H49K, respectively, at various concentrations of proteins. (A) 1 µM (light blue), 3 µM (medium blue), and 9 µM (dark blue); (B) 1 µM (pink) and 10 µM (red). (C and D) Representative fittings of data from chemical denaturation at pH 7.0 of IF₁-Y33W (3 µM; blue) and IF₁-Y33W-H49K (1 µM; pink), respectively. Fitted curves are depicted in black, and the colored dots are experimental values.

Fig. 3.

Effect of pH on dimerization and tetramerization of IF₁. (A) Values of K_d for IF₁-Y33W and IF₁-Y33W-H49K at pH 6.0, 7.0, and 8.0 obtained from chemical denaturation experiments. (B) Simulation of fractional oligomeric distribution for IF₁-Y33W at pH 6.0 (blue), pH 7.0 (orange), and pH 8.0 (gray). The monomeric, dimeric, and tetrameric species are indicated as dashed, solid with filled areas beneath, and solid lines, respectively.

Fig. 4.

Effect of ionic strength and cation-type on the oligomerization of IF₁. (A and B) Thermal denaturation of IF₁-Y33W and IF₁-Y33W-H49K, respectively. Unfolding was assessed by monitoring the change in the MRE value at 222 nm as a function of temperature. Samples were at protein concentrations of 3 µM in 10 mM MOPS, pH 7.0, containing chloride salts of the indicated cations (total ionic strength of 1 M); gray, MOPS alone (total ionic strength 4 mM); K⁺, blue; Na⁺, black; Li⁺, green; Mg²⁺, orange; Ca²⁺, maroon. (C) Fold changes in K_d values compared with condition with no salt. The values were obtained from fitting chemical denaturation data. The ionic strength corresponding to each condition is indicated next to the ion.