Atomistic View on Substrate Transport in an ABC Exporter
Final Report Abstract
The main aim of the project was to study the molecular mechanisms underlying the substrate translocation of membrane transporters by means of molecular dynamics (MD) simulations, in collaboration with experimental partners. The first part of the project focused on a model ATP-binding cassette (ABC) exporter, TM287/288. Using extensive all-atom MD simulations on the multimicrosecond time scale, we could show in atomic-level detail how the transport substrate molecules daunorubicin and verapamil are translocated through the ABC exporter by following the large-scale conformational transition of the transporter from an inward-facing to an outward-facing state. Distinct differences in hydration of the hydrophobic substrate and of the transmembrane cavity in the inward-facing and outward-facing conformations can explain why substrate reuptake is prevented. In addition to TM287/288, we also investigated the ABC transporters MsbA and P-glycoprotein. For MsbA, we found that the disputed wide-open inward-facing conformation is populated in E. Coli cells. MD simulations showed that the wide opening of the lateral portals in this conformation lowers the activation barrier for binding of the transport substrate core lipid A to the main transmembrane cavity of MsbA, which is a key step in the transport mechanism and thus might define a role of the wide-open conformation in the functional working cycle of this transporter. For human P-glycoprotein, a multidrug efflux pump that is overexpressed in many cancer cells and associated with multidrug resistance, we investigated the conformational ensembles of two previously characterised inward-open conformations that differ in the conformation of two key transmembrane helices, TM4 and TM10, which adopt straight and kinked conformations in high-resolution structures solved in detergent and in nanodiscs, respectively. The MD simulations reveal differences between the conformational ensembles of the two conformers in terms of the accessibility of the main cavity of P-glycoprotein to substrate molecules. Furthermore, the simulations show a spontaneous partial conformational transition from a straight to a kinked conformation of one of the helices, suggesting that both conformations can be populated under physiological conditions in a native membrane environment. From a functional perspective, the results indicate that the straight conformer precedes precedes the kinked conformer in the working transport cycle of P-glycoprotein, with the latter conformation being a post substrate-bound state that prevents substrate disengagement. Finally, we used MD simulations to study the loading of a triacylglyceride (TAG) molecule from the inner cavity of a homologue of the MFS transporter Rv1410 from M. tuberculosis into the periplasmic binding protein LprG. The models of the transporter/LprG complex showed that the unusual periplasmic transmembrane helix extensions of the transporter form the binding site for LprG. TAG molecules that were initially positioned inside the main transmembrane cavity of the transporter were observed to spontaneously move along these TM helix extensions into the hydrophobic binding pocket of LprG during the simulations, thus providing a direct visualization of the process. Taken together, this project showed for a number of case examples how MD simulations and experiments can be combined to investigate the functional mechanisms of membrane transporters in terms of the underlying structure and dynamics, at a level beyond what is possible with any of the individual methods alone.
Publications
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Capturing Substrate Translocation in an ABC Exporter at the Atomic Level. Journal of the American Chemical Society, 142(29), 12791-12801.
Göddeke, Hendrik & Schäfer, Lars V.
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Atomistic Dynamics of Alternating Access Mechanism of an ABC Transporter. High Performance Computing in Science and Engineering '19, 117-124. Springer International Publishing.
Göddeke, Hendrik & Schäfer, Lars
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The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells. Science Advances, 8(41).
Galazzo, Laura; Meier, Gianmarco; Januliene, Dovile; Parey, Kristian; De Vecchis, Dario; Striednig, Bianca; Hilbi, Hubert; Schäfer, Lars V.; Kuprov, Ilya; Moeller, Arne; Bordignon, Enrica & Seeger, Markus A.
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Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase. International Journal of Molecular Sciences, 24(17), 13106.
Paweletz, Laura C.; Holtbrügge, Simon L.; Löb, Malina; De Vecchis, Dario; Schäfer, Lars V.; Günther, Pomorski Thomas & Justesen, Bo Højen
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Structural basis for triacylglyceride extraction from mycobacterial inner membrane by MFS transporter Rv1410. Nature Communications, 14(1).
Remm, Sille; De Vecchis, Dario; Schöppe, Jendrik; Hutter, Cedric A. J.; Gonda, Imre; Hohl, Michael; Newstead, Simon; Schäfer, Lars V. & Seeger, Markus A.
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Coupling the role of lipids to the conformational dynamics of the ABC transporter P-gp.
Vecchis, Dario De & Schäfer, Lars V.
