Mechanistic picture for conformational transition of a membrane transporter at atomic resolution

Abstract

During their transport cycle, ATP-binding cassette (ABC) transporters undergo large-scale conformational changes between inward- and outward-facing states. Using an approach based on designing system-specific reaction coordinates and using nonequilibrium work relations, we have performed extensive all-atom molecular dynamics simulations in the presence of explicit membrane/solvent to sample a large number of mechanistically distinct pathways for the conformational transition of MsbA, a bacterial ABC exporter whose structure has been solved in multiple functional states. The computational approach developed here is based on (i) extensive exploration of system-specific biasing protocols (e.g., using collective variables designed based on available low-resolution crystal structures) and (ii) using nonequilibrium work relations for comparing the relevance of the transition pathways. The most relevant transition pathway identified using this approach involves several distinct stages reflecting the complex nature of the structural changes associated with the function of the protein. The opening of the cytoplasmic gate during the outward- to inward-facing transition of apo MsbA is found to be disfavored when the periplasmic gate is open and facilitated by a twisting motion of the nucleotide-binding domains that involves a dramatic change in their relative orientation. These results highlight the cooperativity between the transmembrane and the nucleotide-binding domains in the conformational transition of ABC exporters. The approach introduced here provides a framework to study large-scale conformational changes of other membrane transporters whose computational investigation at an atomic resolution may not be currently feasible using conventional methods.

Keywords: bias-exchange umbrella sampling; conformational free energy; orientation quaternion.

Fig. 1.

Definitions of reaction coordinates. (A) Cartoon representation of MsbA structure in three different conformations: OF (Left), IF-c (Center) (two views), and IF-o (Right) along with the definitions of reaction coordinates α, β, and γ. is colored yellow/green, and TMD bundles B₁ (, helices), B₂ (,), B₃ , and B₄ are colored blue, red, yellow, and green, respectively. In the OF conformation, the roll axes of bundles B₁/B₄ and B₂/B₃ are colored blue and red, respectively, to illustrate the definition of β. In the IF-o conformation, the roll axes of bundles B₁/B₃ and B₂/B₄ are colored blue and red, respectively, to illustrate the definition of α. The roll axes of in the OF and IF-c conformations are colored yellow/green to illustrate the definition of γ. (B) Side (Top and Middle) and top (Bottom) views of the NBDs in OF and IF-c conformations along with the definitions of (Top) and γ (Middle).

Fig. 2.

Three-dimensional reaction coordinate space (α, β, γ). (A) Projection of 20 select trajectories (out of ∼250) onto the (α, β, γ) space along with their projections onto the 2D spaces (α, β), (α, γ), and (β, γ) (all trajectories are shown in SI Appendix, Fig. S1). (B) The smoothed trace of the 160-ns optimized pathway (black) compared with the 160-ns targeted MD (red) pathway. OF (cube), IF-c (sphere), and IF-o (pyramid) crystal structures are also shown in the (α, β, γ) space whose 2D projections are given by square, circle, and triangle, respectively.

Fig. 3.

Nonequilibrium work analysis. (A) Work profiles of all 160-ns trajectories in which the OF → IF-o transition has been induced by steering the system along α, β, and γ (and in some cases d_NBD) in different orders. Color coding is as follows: (i) α → β class (red); (ii) β → α → γ class (orange); (iii) (β, γ ) → α class (blue); the optimized trajectory is colored black, although it belongs to class iii. (B–E) Reconstructed work profiles in α, γ, β, and spaces. Dashed lines are those starting from the OF conformation; (α, β, γ, d_NBD) vectors show the initial state of each simulation; highlighted are the reaction coordinates with a major change before the simulation.

Fig. 4.

Transition pathway of MsbA from the optimum protocol. Snapshots of MsbA structure (in surface representation) along the optimized OF → IF transition pathway (two perpendicular side views). The reaction coordinates and NBD/TMD conformational changes associated with each stage are given. Also see Movie S1 and SI Appendix, Table S4.

Fig. 5.

PMF of apo MsbA along α in the IF state. The PMF is obtained from BEUS MD simulations, and the error bars are estimated using a bootstrapping algorithm (SI Appendix). The MsbA conformations shown (in surface representation) represent the IF-c and IF-o crystal structures (left and right, respectively), as well as an IF conformation (center) that resembles the P-gp crystal structures (–10). The values of α associated with the IF-c and IF-o crystal structures are marked by a circle and a triangle, respectively.