Nucleotide Binding, ATP Hydrolysis, and Mutation of the Catalytic Carboxylates of Human P-Glycoprotein Cause Distinct Conformational Changes in the Transmembrane Segments†

Biochemistry ◽  
2007 ◽  
Vol 46 (32) ◽  
pp. 9328-9336 ◽  
Author(s):  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Transmembrane Segments ◽  
P Glycoprotein

scholarly journals Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter

2006 ◽  
Vol 399 (2) ◽  
pp. 315-323 ◽  
Author(s):  
Paula L. Russell ◽  
Frances J. Sharom
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Functional Characterization ◽  
Tryptophan Fluorescence ◽  
Fold Increase ◽  
Nucleotide Binding ◽  
Forward Direction ◽  
Multidrug Transporter ◽  
Bivalent Cation ◽  
P Glycoprotein

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (Vi) and fluoroaluminate (AlFx) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp–M2+–ADP–X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeFx) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and Vi, BeFx or AlFx using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with Vi and ATP led to a 6-fold increase in the acrylamide quenching constant, KSV, suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with Vi and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)–ATP/TNP–ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeFx-trapped complex was much more polar than that of the Vi and AlFx complexes. Functionally, all the trapped complexes were able to bind drugs and TNP–nucleotides with unchanged affinity compared with native Pgp.

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 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 Characterization of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein

2001 ◽  
Vol 276 (15) ◽  
pp. 11653-11661 ◽  
Author(s):  
Zuben E. Sauna ◽  
Suresh V. Ambudkar
Keyword(s):  
Multidrug Resistance ◽  
Functional Outcomes ◽  
Atp Hydrolysis ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Catalytic Cycle ◽  
Nucleotide Binding Sites ◽  
P Glycoprotein ◽  
Random Manner

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis à visthe substrate, they show comparablet12for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

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.

A molecular understanding of the catalytic cycle of the nucleotide-binding domain of the ABC transporter HlyB

2005 ◽  
Vol 33 (5) ◽  
pp. 990-995 ◽  
Author(s):  
J. Zaitseva ◽  
S. Jenewein ◽  
C. Oswald ◽  
T. Jumpertz ◽  
I.B. Holland ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Central Element ◽  
Nucleotide Binding ◽  
Single Step ◽  
Catalytic Cycle ◽  
Type I ◽  
Individual Crystal

The ABC transporter (ATP-binding-cassette transporter) HlyB (haemolysin B) is the central element of a type I secretion machinery, dedicated to the secretion of the toxin HlyA in Escherichia coli. In addition to the ABC transporter, two other indispensable elements are necessary for the secretion of the toxin across two membranes in a single step: the transenvelope protein HlyD and the outer membrane protein TolC. Despite the fact that the hydrolysis of ATP by HlyB fuels secretion of HlyA, the essential features of the underlying transport mechanism remain an enigma. Similar to all other ABC transporters, ranging from bacteria to man, HlyB is composed of two NBDs (nucleotide-binding domains) and two transmembrane domains. Here we summarize our detailed biochemical, biophysical and structural studies aimed at an understanding of the molecular principles of how ATP-hydrolysis is coupled to energy transduction, including the conformational changes occurring during the catalytic cycle, leading to substrate transport. We have obtained individual crystal structures for each single ground state of the catalytic cycle. From these and other biochemical and mutational studies, we shall provide a detailed molecular picture of the steps governing intramolecular communication and the utilization of chemical energy, due to ATP hydrolysis, in relation to resulting structural changes within the NBD. These data will be summarized in a general model to explain how these molecular machines achieve translocation of molecules across biological membranes.

scholarly journals Demonstration of Conformational Changes Associated with Activation of the Maltose Transport Complex

2001 ◽  
Vol 276 (15) ◽  
pp. 12362-12368 ◽  
Author(s):  
Daynene E. Mannering ◽  
Susan Sharma ◽  
Amy L. Davidson
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Wild Type ◽  
Nucleotide Binding Sites ◽  
Atp Binding Cassette Transporter ◽  
Aqueous Solvent ◽  
In The Wild ◽  
Transport Complex

InEscherichia coli, interaction of a periplasmic maltose-binding protein with a membrane-associated ATP-binding cassette transporter stimulates ATP hydrolysis, resulting in translocation of maltose into the cell. The maltose transporter contains two transmembrane subunits, MalF and MalG, and two copies of a nucleotide-hydrolyzing subunit, MalK. Mutant transport complexes that function in the absence of binding protein are thought to be stabilized in an ATPase-active conformation. To probe the conformation of the nucleotide-binding site and to gain an understanding of the nature of the conformational changes that lead to activation, cysteine 40 within the Walker A motif of the MalK subunit was modified by the fluorophore 2-(4′-maleimidoanilino)naphthalene-6-sulfonic acid. Fluorescence differences indicated that residues involved in nucleotide binding were less accessible to aqueous solvent in the binding protein independent transporter than in the wild-type transporter. Similar differences in fluorescence were seen when a vanadate-trapped transition state conformation was compared with the ground state in the wild-type transporter. Our results and recent crystal structures are consistent with a model in which activation of ATPase activity is associated with conformational changes that bring the two MalK subunits closer together, completing the nucleotide-binding sites and burying ATP in the interface.

scholarly journals Kinesin: switch I & II and the motor mechanism

2002 ◽  
Vol 115 (1) ◽  
pp. 15-23 ◽  
Author(s):  
F. Jon Kull ◽  
Sharyn A. Endow
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Structural Transitions ◽  
Rotational Movement ◽  
Atomic Models ◽  
Binding Cleft

New crystal structures of the kinesin motors differ from previously described motor-ADP atomic models, showing striking changes both in the switch I region near the nucleotide-binding cleft and in the switch II or ‘relay’ helix at the filament-binding face of the motor. The switch I region, present as a short helix flanked by two loops in previous motor-ADP structures, rearranges into a pseudo-β-hairpin or is completely disordered with melted helices to either side of the disordered switch I loop. The relay helix undergoes a rotational movement coupled to a translation that differs from the piston-like movement of the relay helix observed in myosin. The changes observed in the crystal structures are interpreted to represent structural transitions that occur in the kinesin motors during the ATP hydrolysis cycle. The movements of switch I residues disrupt the water-mediated coordination of the bound Mg2+, which could result in loss of Mg2+ and ADP, raising the intriguing possibility that disruption of the switch I region leads to release of nucleotide by the kinesins. None of the new structures is a true motor-ATP state, however, probably because conformational changes at the active site of the kinesins require interactions with microtubules to stabilize the movements.

Sign in / Sign up

Export Citation Format

Share Document