Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier

Abstract

The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.

Keywords: AAC, ADP/ADP carrier; ATP translocases; CATR, carboxyatractyloside; CoA, coenzyme A; GDC, Graves disease carrier protein, or SLC25A16; MCF, mitochondrial carrier family; MD simulation, molecular dynamics simulation PCA, Principal component analysis; Mitochondrial ADP; OXPHOS, oxidative phosphorylation; SCaMCs, short Ca2+-binding mitochondrial carrier, or Mg-ATP/Pi carrier; Solute carrier family 25, molecular dynamics simulation; Substrate recognition; Transporter; c-state, cytosol-open state; m-state, matrix-open state.

Graphical abstract

Fig. 1

A scheme of AAC function and the workflows of this study. (A) A schematic diagram to show the function of AAC. The structures of AAC in c-state (PDB entry: 1OKC) and m-state (PDB entry: 6GCI) are shown. Residues of the previously proposed central binding site (K22, R79, G182, I183 and R279) are highlighted in blue spheres. (B) Workflow of the current work. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Relaxed conformations of c-state apo-AAC are obtained from three 3-μs MD simulations. (A) Different shapes of the pocket entrance in the crystal structure and in the structure at the end of the simulation traj-1. (B) Time evolution of the pocket volume of AAC in the three trajectories. (C) The first component of the PCA analysis on the three trajectories. (D) The difference of the salt bridges near the entrance of AAC in the crystal structure and apo-AAC simulations. Occupancies of the salt bridges are averaged over three trajectories and are shown in red numbers (in percentage). The occupancies of these salt bridges in each trajectory are listed in Table S1.

Fig. 3

Initial ADP binding revealed by large-scale short MD simulations. (A) The movement of ADP phosphate moiety between the positively charged residues near the entrance of carrier in 31 10-ns MD simulations (in S19 and S26 ADP left the carrier in the beginning). The moving direction of each step is shown in gradient line from blue to red. The number of the simulations in which the first contact is observed is provided within the parentheses. (B) The ADP binding occupancies calculated on the 33 10-ns simulation trajectories are shown in colored scale on the 3D structure of c-state AAC. All positively charged residues are highlighted in spheres. (C) The trajectory of the ADP α-phosphate group in one 200-ns extended simulation (e-S33). The simulation time is shown in colored spectrum along the moving trajectory. Initial and ending positions of the ADP α-phosphate group are highlighted in blue and red spheres respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Long-time MD simulations identify a highly specific ADP binding site S2. (A) The highly specific ADP binding mode with site S2. Interaction occupancies are shown in red numbers. The favorable stacking structure is highlighted in the inset, with the bound sodium ion shown with a green sphere. (B) The consensus pathway of ADP locating to the specific binding mode at site S2 in ADP-2 and ADP-3. (C) Free energy landscape built from the χ angle and the z-component of the N6 Cartesian coordinates of ADP in ADP-2. (D) The non-specific ATP binding to the site S2. The favorable stacking to Y194 is highlighted in the inset, with the bound sodium ion shown in a green sphere. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

An intermediate conformation of ADP binding with the central binding site S1 and R187 of site S2. (A) A stable intermediate ADP conformation bridging site S2 and site S1. Residues of site S2 and site S1 are shown in yellow and violet spheres respectively, and ADP is shown in green sticks. (B) The salt bridge network formed between the phosphate moiety of ADP and the positively charged residues of site S1. (C) Conformations of R187 in different structures. (D) Superposition of the snapshots at the end of traj-2 (silver) and ADP-mod (violet). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Adenine nucleotide transporters use the variable positions at the upper region of the cavity to discriminate substrates. (A) AACs fall into the same clade with SLC25A43, SLC25A42, GDC, SCaMCs and SLC25A41 in phylogeny analysis. (B) Sequence logo presentation of the variable positions among adenine nucleotide transporters and their corresponding substrates. Only variable positions that face the central cavity and involve charged residues in at least one of the five transporters are shown. Residues belonging to the specific ADP binding site S2 of AAC are highlighted with red arrows. The residues are numbered based on hAAC1, hSLC25A42, hGDC, hSCaMC1, hSLC25A41 respectively. Complete sequence logos on the odd-numbered and even-numbered helices are provided in Figs. S12, S13 respectively. (C) Mapping the variable positions (shown in spheres) on the c-state structure of AAC.

Fig. 7

The proposed mechanism of ADP recognition and transport in AAC. ADP is shown in green, with the adenine base, ribose ring and phosphate moiety represented with rectangle, pentagon and circle respectively. The specific site S2 is shown in yellow and the central site S1 is shown in violet. The first basic patch is shown in cyan. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)