Abstract
P-glycoprotein (ABCB1) is an important component of barrier tissues that extrudes a wide range of chemically unrelated compounds. ABCB1 consists of two transmembrane domains forming the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that provide the energy by binding and hydrolyzing ATP. We analyzed the mechanistic and energetic properties of the NBD dimer via molecular dynamics simulations. We find that MgATP stabilizes the NBD dimer through strong attractive forces by serving as an interaction hub. The irreversible ATP hydrolysis step converts the chemical energy stored in the phosphate bonds of ATP into potential energy. Following ATP hydrolysis, interactions between the NBDs and the ATP hydrolysis products MgADP + Pi remain strong, mainly because Mg2+ forms stabilizing interactions with ADP and Pi. Despite these stabilizing interactions MgADP + Pi are unable to hold the dimer together, which becomes separated by avid interactions of MgADP + Pi with water. ATP binding to the open NBDs and ATP hydrolysis in the closed NBD dimer represent two steps of energy input, each leading to the formation of a high energy state. Relaxation from these high energy states occurs through conformational changes that push ABCB1 through the transport cycle.
Highlights
ABCB1 (P-glycoprotein) is responsible for multidrug resistance in cancer cells by preventing drugs from reaching the cytosol, thereby leading to failure of chemotherapy treatment[1]
The energetic profile is convoluted by several factors, and the majority of the energy output of the nucleotide binding domains (NBDs) is immediately consumed by the transmembrane domains (TMDs)
We created an isolated NBD system, which allows for separating the energetic contributions of the NBDs from those of the TMDs with the aim to quantify the maximal energy output produced by the NBDs and derive the free energy profile associated with NBD movement
Summary
ABCB1 (P-glycoprotein) is responsible for multidrug resistance in cancer cells by preventing drugs from reaching the cytosol, thereby leading to failure of chemotherapy treatment[1]. The NBDs, the most conserved region of ABC proteins, form at their interface two symmetry-related nucleotide binding sites (NBSs) that provide the energy for substrate transport through ATP binding and hydrolysis. The A-loop and the Walker A motif have a major role in nucleotide binding, the Walker B motif and the H-loop[22] play a direct role in ATP hydrolysis, and the Q-loop[23], X-loop[21] and D-loop[24] are responsible for interdomain communication. Biochemical and structural data have identified conformational changes of the NBD dimer in response to ATP binding and hydrolysis. Elucidation of the driving forces that induce conformational changes and the energy barriers separating states is required for a full understanding of the mechanism by which ATP energizes the transport cycle of ABCB1. PMF profiles allow for quantifying the energy output produced by the NBDs, revealing the maximum energy that can be harvested by ABCB1 for substrate transport
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