Insights into the linker domain in ABCB1/P-glycoprotein

Abstract

ABCB1/P-glycoprotein(P-gp) is one of the best-known ABC transporters and its involvement in the clearance of chemotherapy agents in cancer cells can lead to multidrug resistance. The N- and C-terminal halves of P-gp are joined by a flexible linker region. Linker regions in ABC transporters often play pivotal roles (i.e. CFTR), however, there is no consensus on the importance of the role of the linker, the complete 3D-structure of which has not yet been determined. Prior studies on linker length showed major effects on P-gp function. In this work, we combined microsecond simulations ({approx}50{micro}s) on mouse and human P-glycoprotein, cryo-EM and other computational methods to trace a profile of the linker region. Different experimentally verified conditions involving the linker region were simulated, analysed and compared. Attachment of the linker to the membrane was spontaneously and occasionally encountered via a recurrent series of amino-acids, despite random starting conformations, suggesting that the lipid bilayer might be involved in linker regulation. As carried out in previous experiments, shortening the linker could potentially disconnect the ATPase activity from the substrate transport by interfering with one ATP site while introducing a short partially-structured linker shows remarkable stability in accelerated MD. Finally, we investigated the linker region by positioning it in specific quadrants of the 3D space, with simulations suggesting 1) a possible influence on protein flexibility, 2) selective tightening and opening of the two halves of P-gp, and a 3) possible stabilization of the inward-facing/occluded-state. The interaction between the linker and ATPs was investigated using protein-protein docking, simulations and a re-refined cryo-EM map where medium-low resolution and weak Coulomb density was found between the nucleotide binding domains.