Conversion of chemical to mechanical energy by the nucleotide binding domains of ABCB1

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.

Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion

“…We used MD simulations to probe into the molecular origin of the restored transport function. We previously showed for wild-type ABCB1 that ATP binding stabilizes the NBD dimer by~42 kJ/mol and that the hydrolysis products ADP and inorganic phosphate create a high energy state that induces NBD separation [67,68]. Our simulations revealed that binding of ATP and its interactions with NBS1 are similar for wild-type ABCB1 and the catalytic glutamate mutant, but showed more restricted dynamics in the E556M mutant, indicating stronger interactions that are consistent with a locked state.…”

“…Transporters were stable in all simulations, maintaining their secondary and tertiary structures and showing similar deviations from their respective starting conformations. Detailed analysis showed that the Pγ of ATP interacts with the Mg 2+ ion, the Walker A motif and the signature sequence, while the Pα and Pβ almost exclusively interact with the Walker A motif (Fig 6) [67,68]. Histograms of distances measured between the Cα atom of S434 (first turn of the Walker A helix) and the Pα of ATP showed equally stable interactions in the three variants, with a similar degree of fluctuations ( Fig 6A).…”

“…Our simulations revealed that binding of ATP and its interactions with NBS1 are similar for wild-type ABCB1 and the catalytic glutamate mutant, but showed more restricted dynamics in the E556M mutant, indicating stronger interactions that are consistent with a locked state. ATP cannot be efficiently released, because the free energy of ATP binding shows a deep energy well that stabilizes the ATP-bound conformation [67,68]. Therefore, ATP remains tightly bound in the E556M ABCB1 variant, as efficient hydrolysis is prevented, thereby leading to an arrest of the transport cycle.…”

“…( D ) The matrix summarize all contacts across the NBD-dimer interface, indicating in red the residues mutated in the quadruple mutant. ATP acts as glue [ 21 , 67 ] and interaction hub, while shielding residues from direct interaction.…”

The role of the degenerate nucleotide binding site in type I ABC exporters

“…ATP hydrolysis is asymmetric in ABC transporters with a degenerate site, leading to a high energy state [44,45] in the canonical NBS2 (MgADP + Pi) after hydrolysis of ATP, while degenerate NBS1 contains the nonhydrolyzed ATP. Accordingly, NBD separation will most likely be asymmetric, as MgATP bound to NBS1 represents a low energy state [44,45] that stabilizes the closed NBS1 and thus glues the NBDs together. The motions of NBD separation could in theory still be coordinated and symmetric, if other parts of the transporter would enforce such symmetric motion, similar to hemoglobin, which seems to operate using concerted motion of all four hemoglobin subunits in response to oxygen binding [46].…”