Negative Stain Single-particle EM of the Maltose Transporter in Nanodiscs Reveals Asymmetric Closure of MalK2 and Catalytic Roles of ATP, MalE, and Maltose

Abstract

The Escherichia coli MalE-MalFGK₂ complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK₂; and a periplasmic maltose receptor, MalE. Opening and closure of MalK₂ is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK₂ in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15-25 Å resolution) indicate that binding of ATP to MalK₂ promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK₂ In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK₂ remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.

Keywords: ABC transporter; electron microscopy (EM); membrane protein; membrane transporter reconstitution; single-particle analysis; translocation; transport.

FIGURE 1.

EM map of MalFGK₂ reconstituted in nanodiscs in the absence of nucleotide. A, field of view of particles stained with uranyl formate. A few individual particles are boxed. B, three characteristic views of MalFGK₂ obtained by 2D averaging. Yellow arrowheads point to MalK₂; red arrowheads point to nanodisc; and blue arrowheads point to the P2-loop. C, corresponding projection of the model. D–H, EM map (transparent gray isosurface representation) shown with the docked crystal structure of MalFGK₂ in the resting state (PDB code 3FH6) and the complete MalF P2-loop (isolated from PDB code 2R6G). The color code of MalFGK₂ subunits are used throughout the manuscript: MalK dimer (yellow), MalF (blue), MalG (purple), and MalF P2 loop (green). The residues composing the nucleotide binding site are shown in sphere representation and colored in red (Walker A) and green (LSGGQ). The red arrow points to the coupling helix docked into a surface cleft on each MalK monomer.

FIGURE 2.

Effect of nucleotide on MalFGK₂ conformations. EM maps of MalFGK2 obtained in the absence of nucleotide and presence of various nucleotides as indicated. All maps were obtained from wild type MalF, MalG, and MalK proteins except for the map labeled ATP-BMOE(*). This map was obtained using the variant MalK_S83C stabilized in the closed conformation with ATP and the cross-linker BMOE. The maps are shown as isosurface representation at the contours of 350 and 250 ų (first and second rows, respectively). The crystal structure of MalFGK₂ (PDB code 3FH6) docked in the EM map is shown for orientation purposes. The Walker A and LSGGQ motifs are shown in sphere representation in A, and their positions are marked by green and red triangles, respectively, in B–E. The red dashed line indicates the position used for slicing the maps to show the MalK_F and MalK_G junctions. The distance between the center of mass of the junctions is indicated. Junction distances less than 20 Å and greater than 50 Å are characteristic of the closed and open conformations respectively. Junction distances between 20 and 40 Å were observed for the semi-closed and asymmetric conformations.

FIGURE 3.

The open, closed, and semi-closed symmetric conformations of MalK₂. EM maps of MalFGK₂ maps shown as isosurface representations with the corresponding crystal structure of MalK₂ dimer (upper panels, with PDB code 1Q1E, B; PDB code 2AWO) and ATP (C; PDB code 1Q12). Residues of the Walker A and LSGGQ motifs are shown as red and green spheres, respectively. The lower panels represent top view cross-sections after removal of the densities and atoms above the dashed line.

FIGURE 4.

EM maps of MalE·MalFGK₂ with different nucleotide and maltose. A–F, the EM maps are shown at two thresholds (450 and 350 A³; first and second rows, respectively) in the conditions indicated. The red dashed line indicates the position used for slicing the maps to show the MalK_F and MalK_G junctions (colored red) in the third row. The distance between the center of mass of the junctions is indicated. G, gallery of single particles of the cross-linked MalFGK₂-MalE complexes upon the addition of maltose is shown. The red triangles point to the detached MalE.

FIGURE 5.

Isolation of MalE-x-MalFGK₂. A, SDS-PAGE analysis of MalF_S205CGK₂ (lane 1), MalE (lane 2), and MalF_S205CGK₂ with MalE (lane 3), all incubated with the oxidizing agent copper phenanthroline. B, Superdex 200 gel filtration profile of MalF_S205C GK₂-x-MalE formed by disulfide cross-linking (upper panel) and SDS-PAGE gel of corresponding fractions.

FIGURE 6.

Model of the maltose transport cycle. A, ATP induces closure of MalK₂ (EM map MalFGK₂·ATP+BMOE), but this conformation is unstable in the absence of MalE (EM maps MalFGK₂·non-hydrolyzable ATP and ADP-P_i analogs) and reverts to a semi-closed symmetric conformation. B, MalE stabilizes the ATP-induced closed conformation (EM map MalE·MalFGK₂·ADP-VO₄), and therefore ATP cleavage occurs. C, maltose enhances the return of the transporter to inward facing conformation, thereby increasing the rate of ADP and P_i release and consequently increasing the ATP hydrolytic cycle. MalE alone does not modify the open conformation of MalK₂. The ATPase activity indicated is that previously reported (5, 28).