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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.

SUBMITTER: Kopcho N 

PROVIDER: S-EPMC6805939 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Dynamics of ABC Transporter P-glycoprotein in Three Conformational States.

Kopcho Noah N   Chang Geoffrey G   Komives Elizabeth A EA  

Scientific reports 20191022 1


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<sup>+2</sup>-ATP, and outward-facing P-gp bound to Mg<sup>+2</sup>-ADP-VO<sub>4</sub><sup>-3</sup>. Nucleotide affinity was measured with bio-layer interf  ...[more]

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