scholarly journals Disruption of the Unique ABCG-Family NBD:NBD Interface Impacts Both Drug Transport and ATP Hydrolysis

2020 ◽  
Vol 21 (3) ◽  
pp. 759
Author(s):  
Parth Kapoor ◽  
Deborah A. Briggs ◽  
Megan H. Cox ◽  
Ian D. Kerr
Keyword(s):  
Atpase Activity ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Contact Region ◽  
Chemotherapy Resistance ◽  
Dimer Interface ◽  
Binding Domains ◽  
Related Family ◽  
Allosteric Communication ◽  
C Terminus

ABCG2 is one of a triumvirate of human multidrug ATP binding cassette (ABC) transporters that are implicated in the defense of cells and tissues against cytotoxic chemicals, but these transporters can also confer chemotherapy resistance states in oncology. Understanding the mechanism of ABCG2 is thus imperative if we are to be able to counter its deleterious activity. The structure of ABCG2 and its related family members (ABCG5/G8) demonstrated that there were two interfaces between the nucleotide binding domains (NBD). In addition to the canonical ATP “sandwich-dimer” interface, there was a second contact region between residues at the C-terminus of the NBD. We investigated this second interface by making mutations to a series of residues that are in close interaction with the opposite NBD. Mutated ABCG2 isoforms were expressed in human embryonic kidney (HEK) 293T cells and analysed for targeting to the membrane, drug transport, and ATPase activity. Mutations to this second interface had a number of effects on ABCG2, including altered drug specificity, altered drug transport, and, in two mutants, a loss of ATPase activity. The results demonstrate that this region is particularly sensitive to mutation and can impact not only direct, local NBD events (i.e., ATP hydrolysis) but also the allosteric communication to the transmembrane domains and drug transport.

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 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 The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

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 Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels

2015 ◽  
Vol 145 (4) ◽  
pp. 261-283 ◽  
Author(s):  
Luiz A. Poletto Chaves ◽  
David C. Gadsby
Keyword(s):  
Atp Hydrolysis ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Dimer Interface ◽  
Binding Domains ◽  
Channel Opening ◽  
Order Of Magnitude ◽  
Signature Sequence ◽  
Dependent Modification ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.

Actin filaments stimulate the Na(+)-K(+)-ATPase

1995 ◽  
Vol 269 (5) ◽  
pp. F637-F643 ◽  
Author(s):  
H. F. Cantiello
Keyword(s):  
Sequence Analysis ◽  
Atpase Activity ◽  
Actin Filaments ◽  
Functional Role ◽  
Atp Hydrolysis ◽  
Rat Kidney ◽  
Alpha Subunit ◽  
Actin Binding ◽  
Binding Domains

In this report, the functional role of actin on Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity was explored. The Na(+)- and K(+)-dependent, ouabain-sensitive ATP hydrolysis mediated by rat kidney Na(+)-K(+)-ATPase increased by 74% in the presence of previously unpolymerized actin (24 microM), whereas addition of polymerized actin was without effect. Addition of actin was associated instead with an increase in the affinity of the Na(+)-K(+)-ATPase for Na+ but not other enzymatic substates. A maximal stimulatory effect (296%) was observed either at an Na(+)-K(+)-ATPase:actin ratio of 1:50,000 or at lower ratios (1:625) by shifting from the E2 (K+ selective) to the E1 (Na+ selective) conformation of the enzyme. Immunoblotting of actin to the purified Na(+)-K(+)-ATPase suggested that this interaction may be linked to binding of actin to the enzyme, which was further supported by sequence analysis indicating putative actin-binding domains in the alpha-subunit of the enzyme. The interaction between actin and the Na(+)-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport.

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 C-terminus of mitotic centromere-associated kinesin (MCAK) inhibits its lattice-stimulated ATPase activity

2004 ◽  
Vol 383 (2) ◽  
pp. 227-235 ◽  
Author(s):  
Ayana MOORE ◽  
Linda WORDEMAN
Keyword(s):  
Amino Acids ◽  
Atpase Activity ◽  
Energy Cost ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Full Length ◽  
Apparent Affinity ◽  
Tubulin Dimer ◽  
C Terminus

Mitotic centromere-associated kinesin (MCAK) is a microtubule (MT)-destabilizing molecular motor. In the present study we show that the final 8 amino acids of the C-terminus of MCAK inhibit lattice-stimulated ATPase activity of the motor. Surprisingly, loss of this C-terminal ‘tail’ (MCAK-Q710) leads to more rapid depolymerization of MTs relative to full-length MCAK (wt-MCAK). Biochemical and microscopic assays revealed that MCAK-Q710 bound to the MT lattice with higher apparent affinity as compared with wt-MCAK. End-stimulated depolymerization was similar for both enzymes. These data suggest that lattice-bound MCAK can increase the rate of MT depolymerization, but at an energy cost. The function of the C-terminus of MCAK may be to selectively inhibit lattice-stimulated ATPase activity, resulting in limited interactions of the motor with the MT lattice. This increases the coupling between ATP hydrolysis and tubulin dimer release, but it also limits MT depolymerization.

scholarly journals Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Csaba Mihályi ◽  
Beáta Töröcsik ◽  
László Csanády
Keyword(s):  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Nucleotide Binding Domain ◽  
Dimer Interface ◽  
Binding Domains ◽  
Gating Mechanism ◽  
Pore Opening ◽  
The Stability ◽  
Structural Architecture ◽  
Closed Pore

In CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) opens the pore, and dimer disruption following ATP hydrolysis closes it. Spontaneous openings without ATP are rare in wild-type CFTR, but in certain CF mutants constitute the only gating mechanism, stimulated by ivacaftor, a clinically approved CFTR potentiator. The molecular motions underlying spontaneous gating are unclear. Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels. We show that spontaneous openings are also strictly coupled to NBD dimerization, which may therefore occur even without ATP. Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability, but not the fundamental structural architecture, of open- and closed-pore conformations. This explains correlated effects of phosphorylation, mutations, and drugs on ATP-driven and spontaneous activity, providing insights for understanding CF mutation and drug mechanisms.

scholarly journals Chromokinesins NOD and KID Use Distinct ATPase Mechanisms and Microtubule Interactions to Perform a Similar Function

2019 ◽  
Author(s):  
Benjamin C. Walker ◽  
Wolfram Tempel ◽  
Haizhong Zhu ◽  
Heewon Park ◽  
Jared C. Cochran
Keyword(s):  
Cell Division ◽  
Bound States ◽  
Catalytic Domain ◽  
Atp Hydrolysis ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Domain Structures ◽  
Allosteric Communication ◽  
Conventional Kinesin ◽  
Communication Pathway

Chromokinesins NOD and KID have similar DNA binding domains and functions during cell division, while their motor domain sequences show significant variations. It has been unclear whether these motors have similar structure, chemistry, and microtubule interactions necessary to follow a similar mechanism of force mediation. We used biochemical rate measurements, cosedimentation, and structural analysis to investigate the ATPase mechanisms of the NOD and KID core domains. These experiments and analysis revealed that NOD and KID have different ATPase mechanisms, microtubule interactions, and catalytic domain structures. The ATPase cycles of NOD and KID have different rate limiting steps. The ATPase rate of NOD was robustly stimulated by microtubules albeit its microtubule affinity was weakened in all nucleotide bound states. KID bound microtubules tightly in all nucleotide states and remained associated with the microtubule for more than 100 cycles of ATP hydrolysis before dissociating. The structure of KID was most similar to conventional kinesin (KIF5). Key differences in the microtubule binding region and allosteric communication pathway between KID and NOD are consistent with our biochemical data. Our results support the model that NOD and KID utilize distinct mechanistic pathways to achieve the same function during cell division.

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