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Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

ABSTRACT: P-glycoprotein (P-gp) is a highly substrate-promiscuous efflux transporter that plays a critical role in drug disposition. P-gp utilizes ATP hydrolysis by nucleotide-binding domains (NBDs) to drive transitions between inward-facing (IF) conformations that bind drugs and outward-facing (OF) conformations that release them to the extracellular solution. However, the details of the protein dynamics within either macroscopic IF or OF conformation remain uncharacterized, and the functional role of local dynamics has not been determined. In this work we measured the local dynamics of the IF state of P-gp in lipid nanodiscs and in detergent solution by hydrogen-deuterium (H/D) exchange MS. We observed "EX1 exchange kinetics," or bimodal kinetics, for several peptides distributed in both NBDs, particularly for P-gp in the lipid nanodiscs. Remarkably, the EX1 kinetics occurred on several time scales, ranging from seconds to hours, suggesting highly complex, and correlated, motions. The results indicate at least three distinct conformational states in the ligand-free P-gp and suggest a rough conformational landscape. Addition of excess ATP and vanadate, to favor the OF conformations, caused a generalized, but modest, decrease in H/D exchange throughout the NBDs and slowed the EX1 kinetic transitions of several peptides. The functional implications of the results are consistent with the possibility that conformational selection provides a source of substrate promiscuity.

SUBMITTER: Li MJ

PROVIDER: S-EPMC5925813 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

Li Mavis Jiarong MJ Guttman Miklos M Atkins William M WM

The Journal of biological chemistry 20180306 17

P-glycoprotein (P-gp) is a highly substrate-promiscuous efflux transporter that plays a critical role in drug disposition. P-gp utilizes ATP hydrolysis by nucleotide-binding domains (NBDs) to drive transitions between inward-facing (IF) conformations that bind drugs and outward-facing (OF) conformations that release them to the extracellular solution. However, the details of the protein dynamics within either macroscopic IF or OF conformation remain uncharacterized, and the functional role of lo ...[more]

PMID: 29511086

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Project description:Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2), and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2), here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2) were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2)H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2) in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2) reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.

Project description:Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

Dataset Information

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

Publications

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale.

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