scholarly journals Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6006
Author(s):  
Juan Xing ◽  
Shuheng Huang ◽  
Yu Heng ◽  
Hu Mei ◽  
Xianchao Pan
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Molecular Design ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Inhibitor Binding ◽  
Binding Domains ◽  
Allosteric Communication ◽  
P Glycoprotein ◽  
Dynamics Simulations

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

scholarly journals Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer–associated P-glycoprotein during ATP hydrolysis

2017 ◽  
Vol 292 (50) ◽  
pp. 20412-20424 ◽  
Author(s):  
Maria E. Zoghbi ◽  
Leo Mok ◽  
Douglas J. Swartz ◽  
Anukriti Singh ◽  
Gregory A. Fendley ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Domains ◽  
P Glycoprotein

scholarly journals Structure–Function Relationships in the Human P-glycoprotein (ABCB1): Insights from Molecular Dynamics Simulations

2021 ◽  
Vol 23 (1) ◽  
pp. 362
Author(s):  
Liadys Mora Lagares ◽  
Yunierkis Pérez-Castillo ◽  
Nikola Minovski ◽  
Marjana Novič
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Efflux Pump ◽  
Transmembrane Protein ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Md Simulations ◽  
Binding Pocket ◽  
Target Cells ◽  
P Glycoprotein

P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette superfamily of transporters, and it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping compounds out of cells. P-gp contributes to a reduction in toxicity, and has broad substrate specificity. It is involved in the failure of many cancer and antiviral chemotherapies due to the phenomenon of multidrug resistance (MDR), in which the membrane transporter removes chemotherapeutic drugs from target cells. Understanding the details of the ligand–P-gp interaction is therefore critical for the development of drugs that can overcome the MDR phenomenon, for the early identification of P-gp substrates that will help us to obtain a more effective prediction of toxicity, and for the subsequent outdesign of substrate properties if needed. In this work, a series of molecular dynamics (MD) simulations of human P-gp (hP-gp) in an explicit membrane-and-water environment were performed to investigate the effects of binding different compounds on the conformational dynamics of P-gp. The results revealed significant differences in the behaviour of P-gp in the presence of active and non-active compounds within the binding pocket, as different patterns of movement were identified that could be correlated with conformational changes leading to the activation of the translocation mechanism. The predicted ligand–P-gp interactions are in good agreement with the available experimental data, as well as the estimation of the binding-free energies of the studied complexes, demonstrating the validity of the results derived from the MD simulations.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

scholarly journals Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

2020 ◽  
Vol 117 (47) ◽  
pp. 29609-29617
Author(s):  
Andaleeb Sajid ◽  
Sabrina Lusvarghi ◽  
Megumi Murakami ◽  
Eduardo E. Chufan ◽  
Biebele Abel ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cancer Drug ◽  
Binding Domains ◽  
Cancer Drug Resistance ◽  
P Glycoprotein ◽  
Cell Transforming

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

scholarly journals Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

2018 ◽  
Vol 115 (22) ◽  
pp. E5193-E5202 ◽  
Author(s):  
Priscilla S.-W. Yeung ◽  
Megumi Yamashita ◽  
Christopher E. Ing ◽  
Régis Pomès ◽  
Douglas M. Freymann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mutational Analysis ◽  
Channel Gating ◽  
Functional Anatomy ◽  
Transmembrane Domains ◽  
Hydrophobic Residues ◽  
Binding Domains ◽  
Allosteric Interactions ◽  
Crac Channel ◽  
Dynamics Simulations

Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1–4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

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