Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors

Dastvan, Reza; Mishra, Shailendra; Peskova, Yelena; Nakamoto, Robert K.; Mchaourab, Hassane S.

doi:10.1126/science.aav9406

Cited by 121 publications

(159 citation statements)

References 36 publications

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“…It is interesting to note that all three studies highlighted asymmetry between the NBDs of the ABC transporters BmrA and P‐gP. These findings are in line with other spectroscopic studies using on P‐gP using DEER or single‐molecule FRET …”

Section: Hdx‐ms Studies Of Membrane Proteins In Detergent Micellessupporting

confidence: 87%

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Martens

Politis

2020

Protein Science

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Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research.They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High-resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX-MS as it has matured into an essential tool for membrane protein structural biologists. K E Y W O R D Sallosteric coupling, conformational dynamics, GPCRs, hydrogen-deuterium exchange mass spectrometry, integral membrane proteins, transporters Standard: LC: Reverse-phase chromatography on C18 column with prior trapping for desalting. MS: Often Synapt or Xevo-G2 in MS E mode √ Drift-time aligned MS E (HDMS E ) 17 √ LC column with shorter alkyl chain-for example, BEH C4 or C8 13 √ Gradient optimization (8-30%, 8-50%) 15 √ Saw-tooth gradient after peptides elution to prevent carryover 16 Miscellaneous observationsUse of PEG-based detergents (e.g., Triton X-100) complicates peptides identification. Good practice to start with a benchmark condition-for example, locked protein. 16,18,19 Adding TCEP helps but too much (>1 M) reduces spectral quality. 15Note: The tick indicates parameters that have shown improvement compared with the standard conditions.

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Section: Hdx‐ms Studies Of Membrane Proteins In Detergent Micellessupporting

confidence: 87%

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Martens

Politis

2020

Protein Science

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“…This was followed by another addition of 0.1 g/ml Biobeads with 1-hour incubation, after which 0.2 mg/ml Biobeads were added and mixed overnight. The next day, 0.2 mg/ml Biobeads were added and mixed for one hour 28 . The reaction was filtered using a 0.45 µm filter to remove Biobeads.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…Prior to publication of the IF structure, we initiated a systematic Double Electron-Electron Resonance (DEER) [28][29][30][31][32] investigation of PfMATE to map proton-and substrate-dependent conformational changes in a lipid bilayer-like environment and to identify sequence motifs of ion and substrate coupling. For this purpose, an extensive network of spin label pairs was introduced at the extracellular and intracellular sides to interrogate ligand-dependent movements of TM helices.…”

mentioning

confidence: 99%

Sequence and Structural Determinants of Ligand-dependent Alternating Access of a MATE Transporter

Jagessar

Claxton

Mchaourab

2019

Preprint

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MATE transporters are ubiquitous ion-coupled antiporters that extrude structurally-and chemically-dissimilar molecules and have been implicated in conferring multidrug resistance.Here, we integrate Double Electron Electron Resonance (DEER) in conjunction with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of liganddependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus. Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inwardfacing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. PfMATE isomerization between outward-facing and inwardfacing conformations is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.

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“…Nevertheless, more work is needed to determine the physical locations of those drug binding sites in Pgp and the spatial interactions of therapeutic drugs and inhibitors. Similarly, the mechanism by which ATP binding and hydrolysis achieve the conformational changes necessary to expel drugs from the cell is only partially understood [47][48][49][50] .…”

mentioning

confidence: 99%

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

Swartz

Singh

Sok

et al. 2020

Sci Rep

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P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysisdriven drug export in pgp are poorly understood. fluorescence spectroscopy using tryptophans (trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supported in vivo activity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.

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Mechanism of allosteric modulation of P-glycoprotein by transport substrates and inhibitors

Cited by 121 publications

References 36 publications

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

Sequence and Structural Determinants of Ligand-dependent Alternating Access of a MATE Transporter

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

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