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. 2017 Mar 22;3(3):250-258.
doi: 10.1021/acscentsci.7b00068. Epub 2017 Mar 7.

Unidirectional Transport Mechanism in an ATP Dependent Exporter

Yanyan Xu  1 , Anna Seelig  2 , Simon Bernèche  1
Affiliations

Unidirectional Transport Mechanism in an ATP Dependent Exporter

Yanyan Xu et al. ACS Cent Sci. .

Abstract

ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the unidirectionality of transport in ABC exporters and suggest a ratio of one ATP hydrolyzed per transport cycle.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Architecture of ABC exporters. The X-ray structure of Sav1866 (PDB ID: 2HYD) is shown with bound ADP molecules. A: Side views of the whole structure. The two subunits are respectively rendered in blue and red. Membrane boundaries are indicated by dashed lines: upper, extracellular side; lower, intracellular side. ADP is shown in green. B: Sequence alignment showing the conservation of motifs in Sav1866 (Sav) and P-glycoprotein (Pgp, ABCB1). nt, ct: N-terminal and C-terminal side in the heterodimer Pgp (Sav is a homodimer). C: Top view of NBD with motifs highlighted. Two NBDs (subunits A and B) form two nucleotide binding sites, NBS I and NBS II. D: Side view of NBD motifs. The X-loop and the signature motif are connected by a short loop, colored in black. E: Scheme of the two NBSs, highlighting the two binding distances, Sb_Wa and Sa_Wb. Sa, Sb: signature motif from subunit A(a) and B(b). Wa, Wb: Walker A from the two subunits. Sb_Wa: distance between Sb and Wa, at NBS I. Sa_Wb: distance between Sa and Wb, at NBS II.
Figure 2
Figure 2
Two different conformations of the TMDs. A: Outward-open and outward-closed conformation of TMDs, taken at t = 100 ns in the simulations of the ATPI_ADPII and 2ATP states, respectively. Membrane boundaries are indicated by dashed lines. B, C: Volume of the cavity formed by the TMDs at the level of the extracellular leaflet (B) and intracellular leaflet (C) for different nucleotide occupancy states.
Figure 3
Figure 3
ATP hydrolysis disrupts the nucleotide binding site and allows movement of X-loop. A: Time-series of distance between signature motif and Walker A for different nucleotide occupancy states. Sa, Sb: signature motif from subunit A(a) and B(b) (see Figure 1C). Wa, Wb: Walker A from the two subunits. Sb_Wa: distance between Sb and Wa, at NBS I. Sa_Wb: distance between Sa and Wb, at NBS II. See the scheme in Figure 1E. B: Time-series of distances between X-loop and Walker A. Xa, Xb: X-loop from subunit A(a) and B(b) (see Figure 1C). Xb_Wa: distance between Xb and Wa, at NBS I. Xa_Wb: distance between Xa and Wb, at NBS II.
Figure 4
Figure 4
ATP hydrolysis at one binding site influences helix 3 and helix 4 in both subunits. A: Helix 3 and helix 4 in a superposition of snapshots taken at t = 100 ns in simulations of the 2ATP (red) and ATPI_ADPII (blue) states. B: Time-series of the distance between the Q208 backbone center-of-mass of the two subunits. C, D: Close-up view of the bottom of helix 3 and helix 4. (C: 2ATP. D: ATPI_ADPII.) E: Time-series of the distance between G118 and G201.
Figure 5
Figure 5
Kink angle in helix 4 characterizes the outward-closed and outward-open conformations of the TMD. A: Comparison of the helix 3/helix 4 hairpin in the outward-closed (red) and outward-open (blue) conformations. Gly138 and Gly183 allow for a kink in the middle of helix 3 and helix 4, respectively. The orange arrow points toward the TMD central cavity. B, C: Density histogram of the kink angle for different nucleotide occupancy states. (B: θ1 in helix 3. C: θ2 in helix 4.)
Figure 6
Figure 6
A network formed by helix 1, helix 3, helix 4, and helix 6. A: Conformations of the four helices in the outward-open conformation. (Up: side view. Down: top view.) B: Conformations of the four helices in the outward-closed conformation (Up: side view. Down: top view.) Rotation of helix 1 and helix 6 (green arrows) is central to the transition from the outward-open conformation to the outward-closed conformation. The hairpin formed by helix 3 and helix 4 moves together with helix 1 and helix 6 (orange arrows). C: Stable salt-bridges contribute to the mechanical coherence between the different helices. (Left: between helix 4 and helix 6. Middle: between helix 3 and helix 6. Right: between helix 1 and helix 6.) For clarity, helix 1 and helix 6 are highlighted in blue, while helix 3 and helix 4 are in red.
Figure 7
Figure 7
Unidirectional transport cycle in ABC exporters. (1) With two ATP bound to the NBDs, the TMDs are in an outward-closed state. (2) Hydrolysis of ATP and release of the inorganic phosphate (Pi) leads to an asymmetric occupation state of the NBDs, which initiates the opening of the TMDs. (3) The TMDs adopt an outward-open conformation, which allows water molecules to fill in and leaves room for allocrite flopping. (4) The binding of ATP to the empty NBS restores the symmetric occupancy state and favors the outward-closed conformation, which leads the allocrites on the extracellular side of the cavity between the TMDs to be squeezed out to the membrane.
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