P-glycoprotein (Pgp) is an ATP-binding cassette exporter that uses ATP hydrolysis to actively transport diverse substrates across the cellular membrane. ATP hydrolysis takes place in the nucleotide binding domains (NBDs) resulting in conformational transition of the transporter. Many studies suggested two different models for ATP hydrolysis in NBDs, symmetric and asymmetric. In the symmetric model, two ATPs are hydrolyzed at the same time, however, the asymmetric model suggests a two-stroke process corresponding to one ATP hydrolysis at a time. Numerous studies have revealed the importance of a number of conserved structural motifs including several loops in ATP hydrolysis. However, the mechanistic role of conserved loop residues and their protonation states in ATP hydrolysis-initiated transport cycle remain elusive. Here, we used equilibrium MD simulations to study the interaction between conserved loops in the nucleotide binding sites, ATP molecules, and catalytic water during the transport cycle in Pgp. The ATP hydrolysis was simulated by using the alchemical free energy perturbation method. The system was equilibrated before and after the first ATP hydrolysis. Then, the second ATP was hydrolyzed and the system was equilibrated again. Our analysis showed that the interactions between the aspartate-loop (D-loop), histidine-loop (H-loop), and gamma-phosphate of ATP (Pγ) trap the catalytic water in NBD1. D-loop is found to interact more closely with Pγ in NBD1, when compared to NBD2. However, after ATP hydrolysis in NBD1, similar interactions are formed between the conserved loops, catalytic water, and ATP in NBD2 favoring subsequent ATP hydrolysis. Furthermore, we also observed that protonated histidine in H-loop promotes the formation of favorable interactions enabling ATP hydrolysis. These results support an asymmetric ATP hydrolysis in Pgp where the first ATP hydrolysis may be the trigger for the second hydrolysis.

Conformational coupling to asymmetric ATP hydrolysis in the transport cycle of p-glycoprotein

2019 Spring National Meeting

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