Flexibility in the ABC transporter MsbA: Alternating access with a twist

Abstract

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.

Fig. 1.

Stereoviews of three conformations of MsbA. (A) Nucleotide bound. (B) Open apo. (C) Closed apo. One monomer in each model is colored with a rainbow gradient (N terminus is blue, C terminus is red), and the other is white. TM helices (TM1–TM6), extracellular loops (EL1–EL3), and intracellular helices (IH1–IH2) are labeled accordingly. AMPPNP molecules are displayed as blue sticks in the nucleotide-bound structure. In all structures, TM4/TM5/IH2 (yellow and orange) associates with the opposite monomer in a conserved manner.

Fig. 2.

Electron-density maps for MsbA-AMPPNP. (A) Stereoview of a single MsbA-AMPPNP dimer (Cα model) contoured at 1σ (blue ribbon) with solvent-flattened and NCS-averaged density map (gray mesh) derived from MAD experimental phases. The heavy-atom mercury locations (red surface) and corresponding Cα positions of Cys-88 and -315 (yellow balls) are superimposed on the model. For clarity, the mercury positions have been labeled for only one monomer. The unaccounted density is from symmetry-related molecules. (B) Stereoview of side-chain electron density for MsbA-AMPPNP at 3.7-Å resolution derived from an averaging omit map phased from a model excluding nucleotide and truncated to polyalanine/glycine (white mesh, contour 1σ). The stacking of Phe-105 and -230 residues on adjacent TM helices and density for Arg-102, Met-109, Met-210, and His-214 are shown as yellow sticks. The initial phases for the averaging omit map were derived from a full model excluding nucleotide and truncated to polyalanine/glycine. The initial unaveraged map (blue mesh, contour 1σ) using polyalanine/glycine model phases is shown for reference. The recovered densities for the side chains are a consequence of the fourfold NCS averaging; these maps reveal the position of several bulky side chains and confirm the registration of amino acids in the model. (C) Stereoview of the same averaging omit map electron density (white mesh, contour 1σ) showing the positions of Phe-116, His-214, Phe-349, Tyr-351, Arg-360, Phe-392, Tyr-393, and Arg-416 in the NBD as yellow sticks. (D) Stereoview of averaging omit density (white mesh, contour 1σ) for AMPPNP (green, blue, red, orange sticks) and the MsbA-AMPPNP model (white sticks). Tyr-351 is shown as magenta sticks.

Fig. 3.

Electron-density maps for MsbA apo structures. (A) Stereoview of a single MsbA-open-apo dimer (Cα model) contoured at 1σ (blue ribbon) with solvent-flattened and NCS-averaged density map (gray mesh) derived from MAD/SIRAS combined experimental phases. (B) Stereoview of a single MsbA-closed-apo dimer (Cα model) contoured at 1σ (blue ribbon) with solvent-flattened and NCS-averaged density map (gray mesh) derived from MAD experimental phases. The heavy-atom Hg positions (red surface) and corresponding Cα positions (yellow balls) of Cys-88, -315, and -401 are superimposed onto the structure. For clarity, the mercury positions are labeled for only one monomer. The unaccounted density is from symmetry-related molecules.

Fig. 4.

Comparison of the MsbA monomers. (A) Stereoview of the conformational changes within the MsbA monomer demonstrating the mobility of TM4/TM5/IH2. The NBDs have been removed for clarity. Monomers are aligned on TM helices 1, 2, 3, and 6. Two NCS-related monomers of MsbA-open-apo that differ in the relative positioning of TM4/TM5/IH2 are shown in blue and cyan (see also SI Fig. 11). The monomers from MsbA-closed-apo and MsbA-AMPPNP are shown in green and yellow, respectively. IH1 moves very little, whereas TM4/TM5/IH2 moves significantly.

Fig. 5.

Summary of conformational changes in MsbA. (A) Conformational changes within the MsbA dimer alter the accessibility to the internal chamber from inward to outward facing. For clarity, only TM helices (labeled 1–6) of one monomer (cyan) are shown inside a surface rendering of the dimer. The open and closed apo conformations form an inward-facing V between TM4/TM5 and TM3/TM6 (red asterisk). The nucleotide-bound conformation (MsbA-AMPPNP) forms an outward-facing V between TM3/TM6 and TM1/TM2, just above the elbow helix (black asterisk). Upon nucleotide binding, TM4/TM5/IH2 moves, causing TM3/TM6 to split away from TM1/TM2, which results in an outward-facing conformation. Both inward- and outward-facing conformations are mediated by intramolecular interactions within a single monomer, but by different sets of helices. (B) Simplified cartoon model illustrating the points above. The relative position of each TM helix is labeled with a number (one monomer in white and the other in gray). The arrows illustrate the motions required to go to the next state. (C) Top-down view of NBDs (one monomer shown in white and the other in gray). IH1 (green) and IH2 (yellow) from both monomers are shown. In the absence of nucleotide (apo), the NBDs are in similar orientations with the ATP-binding half-sites (LSGGQ and P-loop) facing each other; the P-loops (red) are roughly aligned (dashed lines) with one another across the dimer interface. Upon nucleotide binding (AMPPNP - magenta), the canonical ATP sandwich is formed, aligning the nucleotide between the LSGGQ and P-loop. IH1 tracks with the cis-monomer, whereas IH2 tracks with the trans-monomer. The motion of the NBDs from closed-apo- to nucleotide-bound transmits a structural change (described above) to the TMs via IH1 and IH2, resulting in an outward-facing conformation.