Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

Abstract

We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg⁺²-ATP, and outward-facing P-gp bound to Mg⁺²-ADP-VO₄^-3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg⁺²-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation.

Figure 1

(A) Domain organization of P-gp. HDX-MS data have been mapped on the inward-facing crystal structure of P-gp. The model is colored on a rainbow scale from lowest (blue) to highest (red) by relative deuterium uptake of apo P-gp after 5 minutes of exchange. Solvent occlusion by the detergent micelle is indicated by low exchange along the transmembrane domain. (B) Diagram of the conformational transition between inward and outward-facing states. A homology model based on outward-facing MsbA was used to represent outward-facing P-gp. Structures are colored by difference in relative uptake between inward and outward-facing P-gp after 5 minutes of exchange. Regions with decreased exchange in the outward-facing state appear blue, while regions with greater exchange are colored red.

Figure 2

Binding kinetics resulting from BLI experiments are summarized. Both mutant and wild-type P-gp displayed similar affinities for ATP and AMPPNP. Wild-type P-gp binding to Mg⁺²-ATP in the presence of VO₄⁻³ was approximately 10-fold tighter. Concentrations are 1.6 (black), 8 (blue), 40 (green), 200 (orange), 1000 (red) and 5000 (cyan) µM. The kinetic parameters are listed in the Table below the plots.

Figure 3

Deuterium uptake plots for regions of TM2 and TM8. These helices are centered along the sides of the transporter, and are the only TMHs which displayed low uptake outside the detergent band.

Figure 4

Deuterium uptake plots for the TMHs comprising the polyspecific binding pocket. All regions display increasing uptake over time, indicative of ongoing dynamic processes throughout this region. TM4 (residues 216–223) is the only region within the detergent band that exchanged with deuterium.

Figure 5

Deuterium uptake plots of the regions corresponding to NBD regions contacting ICHs. (A) All regions contacting ICHs from both NBDs decreased exchange in the pre-hydrolytic state. (B) In the outward-facing state, every region contacting ICHs on NBD1 decreased exchange by more than 0.5 Da. Only the peptide containing residues 1123–1138 showed decreased exchange in NBD2.

Figure 6

Deuterium uptake plots of the ICHs. (A) In the pre-hydrolytic state, ICH4 contacting NBD1 and ICH2 contacting NBD2 showed decreased exchange. (B) In the outward-facing state, both ICHs contacting NBD1 showed reduced exchange, while the ICHs at the NBD2 interface did not.

Figure 7

Deuterium uptake plots reveal the dynamics of the EC domain. (A) A cryo-EM structure of (E552Q/E1197Q) P-gp obtained under similar experimental conditions provided the best fit to our HDX-MS data. Decreased exchange was evident among ECL1, 5 and 6 in the pre-hydrolytic state. (B) In outward-facing P-gp, increased exchange was found among ECL 2, 3, 4 and 6.