Simulations of substrate transport in the multidrug transporter EmrD

Abstract

EmrD is a multidrug resistance (MDR) transporter from Escherichia coli, which is involved in the efflux of amphipathic compounds from the cytoplasm, and the first MDR member of the major facilitator superfamily to be crystallized. Molecular dynamics simulation of EmrD in a phospholipid bilayer was used to characterize the conformational dynamics of the protein. Motions that support a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the EmrD central cavity were observed. In addition, the translocation pathway of meta-chloro carbonylcyanide phenylhydrazone (CCCP) was probed using both standard and steered molecular dynamics simulation. In particular, interactions of a few specific residues with CCCP have been identified. Finally, a large motion of two residues, Val 45 and Leu 233, was observed with the passage of CCCP into the periplasmic space, placing a lower bound on the extent of opening required at this end of the protein for substrate transport. Overall, our simulations probe details of the transport pathway, motions of EmrD at an atomic level of detail, and offer new insights into the functioning of MDR transporters.

Figure 1

(a) Initial cytoplasmic view of EmrD from the apo-a simulation with helix numberings and locations of the N-ter (blue) and C-ter (red). (b) Image of EmrD with periplasm and cytoplasm labeled and a ruler indicating approximate z positions along the protein. Change in the separations of the helices between the last 1 ns and the first 1 ns of the (c) apo-a and (d) apo-b simulations. The diagonal elements are all equal to 0, the elements above the diagonal black line are the changes in separation between the cytoplasmic ends of the helices, and the elements below are the changes in separation between the periplasmic ends of the helices.

Figure 2

EmrD conformation at the beginning (a) and the end (b) of the apo-a simulation, with central cavity profiles. Cavity profiles are colored red (radius < 1.15 Å), green (radius between 1.15 Å and 2.30 Å) and blue (radius > 2.30 Å). Residues 252 to 266 are shown as yellow spheres. Residues in the “selectivity filter” are shown as red spheres. The brown spheres represent lipid phosphorous atoms.

Figure 3

Radius of the central cavity of EmrD from the apo simulations. The red solid and dashed curves are an average over trajectory snapshots of the central cavity radius from the first 1 ns and the last 1 ns of the apo-a simulation respectively. The blue solid and dashed curves are the same but for the apo-b simulation. The black solid curve is the x-ray profile. The approximate positions of Tyr 52, Tyr 56, and Arg 118 are indicated.

Figure 4

Conformational changes upon translocation. (a) Cavity profile for the EmrD x-ray structure aligned with the black dashed curve in (b) and (c). Average cavity radius (taken over 1 ns), at several intervals of the simulation, for v1-a (b) and v1-b (c). For each figure the time-evolution goes in order as blue, green, orange and red. The corresponding colored arrows on the vertical axis show the motion of the center of mass of CCCP while each average radius profile is obtained. (d) Cavity profile obtained from HOLE for four representative frames from v1-a corresponding to the blue, green, orange and red curves (left to right, respectively). Cavity profiles are colored red (radius < 1.15 Å), green (radius between 1.15 Å and 2.30 Å) and blue (radius > 2.30 Å). The coloring of helices is the same as in Fig. 1 a.

Figure 5

Interactions of CCCP (vdW and electrostatic) with each of the helices, H1–H12 for both the v1-a (a and b) and v1-b (b and c) simulations as a function of the position of CCCP's center of mass. (a) and (c) show the N-domain helices H1–H6 (black, red, green, blue, orange and violet respectively). (b) and (d) show the C-domain helices H7–H12 (black, red, green, blue, orange and violet respectively). (e) EmrD colored by interaction energy with CCCP. Blue, green, orange and red correspond to average interaction energies greater than 0 kcal/mol, between 0 and −3 kcal/mol, −3 to −6 kcal/mol, and below −6 kcal/mol respectively. Grey coloring means that CCCP never came within a 3 Å cutoff distance from that region of the protein.

Figure 6

Interactions within the central cavity observed in fs-a. Tyr 52 (red), Tyr 56 (blue), Phe 249 (purple), Trp 300 (green), Arg 118 (brown) and CCCP (yellow). (a) nearly parallel ring stacking of the aromatic rings of Tyr 52, Tyr 56 and CCCP. (b) the aromatic ring of CCCP interacting with the side chain of Tyr 56, while the nitrile groups interact with the Arg 118 side chain. Figure (c) shows interaction energies between the five residues pictured and CCCP, with same coloring as in (a) and (b).

Figure 7

Motion of Leu 233 (located on H7–H8 loop, red) and Val 45 (located at periplasmic end of H2, pink) as CCCP (yellow) passes into the periplasmic space observed in v1-a. (a) initial snapshot, and (b) as CCCP is passing out of the central cavity. (c) Distance between the methyl carbon atoms of the side chains of Leu 233 and Val 45 as a function of the z position of the center of mass of CCCP as it moves from the cytoplasmic side (left) to the periplasmic side (right) of EmrD. The black and red curves in (c) correspond to v1-a and v1-b, respectively.