Pathway of binding of the intrinsically disordered mitochondrial inhibitor protein to F1-ATPase

Abstract

The hydrolysis of ATP by the ATP synthase in mitochondria is inhibited by a protein called IF1. Bovine IF1 has 84 amino acids, and its N-terminal inhibitory region is intrinsically disordered. In a known structure of bovine F1-ATPase inhibited with residues 1-60 of IF1, the inhibitory region from residues 1-50 is mainly α-helical and buried deeply at the α(DP)β(DP)-catalytic interface, where it forms extensive interactions with five of the nine subunits of F1-ATPase but mainly with the β(DP)-subunit. As described here, on the basis of two structures of inhibited complexes formed in the presence of large molar excesses of residues 1-60 of IF1 and of a version of IF1 with the mutation K39A, it appears that the intrinsically disordered inhibitory region interacts first with the αEβE-catalytic interface, the most open of the three catalytic interfaces, where the available interactions with the enzyme allow it to form an α-helix from residues 31-49. Then, in response to the hydrolysis of an ATP molecule and the associated partial closure of the interface to the αTPβTP state, the extent of the folded α-helical region of IF1 increases to residues 23-50 as more interactions with the enzyme become possible. Finally, in response to the hydrolysis of a second ATP molecule and a concomitant 120° rotation of the γ-subunit, the interface closes further to the α(DP)β(DP)-state, allowing more interactions to form between the enzyme and IF1. The structure of IF1 now extends to its maximally folded state found in the previously observed inhibited complex.

Keywords: binding site; folding; inhibitory path; rotary catalysis.

Fig. 1.

The structure of the F₁-(I1-60His)₂ and F₁-(I1-60His-K39A)₃ complexes. The α-, β-, and γ-subunits are red, yellow, and dark blue, the nucleotides are black, and the inhibitor proteins I1-60_E, I1-60_TP, and I1-60_DP are purple, pink, and light blue, respectively. (A and B) Side view of the F₁-(I1-60His)₂ and F₁-(I1-60His-K39A)₃ complexes, respectively; (C and D) cross-sectional view in the C-terminal domains of the α- and β-subunits looking along the axis of the coiled-coil region in the γ-subunit away from the membrane domain of the intact F₁F_o-ATPase, showing the three α- and the three β-subunits arranged in alternation around the γ-subunit, with C an inhibitor protein bound at two of the three catalytic interfaces of the F₁-(I1-60His)₂, and D at all three catalytic interfaces of F₁-(I1-60His-K39A)₃.

Fig. 2.

Cross-sectional side views of the central stalk and the β_E-, β_TP-, and β_DP-subunits of the F₁-(I1-60His-K39A)₃ complex. (A–C) Resolved regions of I1-60_E, I1-60_TP, and I1-60_DP, respectively, bound to the C-terminal domains of their respective catalytic β-subunits, and in the case of I1-60_DP, its interactions with the γ-subunit. The β- and γ-subunits are yellow and dark blue, the nucleotides are black, and the inhibitor proteins I1-60_E, I1-60_TP, and I1-60_DP are purple, pink, and light blue, respectively.

Fig. 3.

The structures of I1-60_E, I1-60_TP, I1-60_DP and their interactions with subunits of F₁-ATPase in the F₁-(I1-60His-K39A)₃ complex. (A–C) Respectively, side chains of the resolved regions of I1-60_E, I1-60_TP, and I1-60_DP interacting with amino acid residues in the C-terminal domains of the β_E-, β_TP-, and β_DP- subunits, plus E454, which does not interact directly with IF₁, but its backbone helps to form the hydrophobic pocket surrounding F34. The depicted regions of I1-60_E, I1-60_TP, and I1-60_DP (residues 31–49, 23–50, and 18–50, respectively) are light blue, and the interacting residues in the β_E-, β_TP-, and β_DP-subunits, the γ-subunit in B, and the α_DP-subunit in C are yellow, dark blue, and red, respectively.

Fig. 4.

The folding of the intrinsically disordered IF₁ upon interaction with F₁-ATPase. In solution, the inhibitor is disordered and becomes progressively folded after initial interaction at the α_Eβ_E-catalytic interface (E) of F₁-ATPase, and subsequently the catalytic interface changing to a α_TPβ_TP interface (TP) and finally a α_DPβ_DP interface (DP). (A) The inhibitor residues that are involved in charged interactions with F₁-ATPase. These interactions increase during the binding of the inhibitor leading to an increase in enthalpy that facilitates folding of the IF₁. (B) The inhibitor residues that are in hydrophobic interactions with F₁-ATPase. These interactions increase during the binding of the inhibitor, leading to an increase in entropy of water molecules that were surrounding the unfolded inhibitor. Residues colored yellow, blue, or orange interact with the neighboring β-subunit, γ-subunit, or the closest nonneighboring β-subunit, respectively.