Conformational Transitions Induced by the Binding of MgATP to the Vitamin B12 ATP-binding Cassette (ABC) Transporter BtuCD

Oloo, Eliud O.; Tieleman, D. Peter

doi:10.1074/jbc.m405084200

Cited by 88 publications

(83 citation statements)

References 57 publications

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“…5A (0.22 mM). These results are consistent with recent real time computer simulations of the ATP binding to BtuCD that find only one ATP molecule occluded, which is sufficient to stabilize the dimer in a "tight binding" state (58). A striking feature was observed at the nucleotide binding pockets in that the formation of the dimer at one binding site resulted in the binding pocket opening at the other.…”

Section: For a Review)supporting

confidence: 90%

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

Sauna

Nandigama

Ambudkar

2006

Journal of Biological Chemistry

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The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E⅐S, P-glycoprotein⅐ATP) and enzyme-product (E⅐P, P-glycoprotein⅐ ADP⅐P i ) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[␣-32 P]ATP as well as under equilibrium conditions using [␣-32 P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[␣-32 P]ATP or [␣-32 P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E⅐S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E⅐P state continues to have low affinity for substrate, suggesting that conformational changes that form the E⅐S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E⅐S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

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Section: For a Review)supporting

confidence: 90%

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

Sauna

Nandigama

Ambudkar

2006

Journal of Biological Chemistry

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“…The first is an open dimer, the second is a closed dimer that is symmetrical with ATPs bound at both NBDs, and the third is the asymmetrical occluded state where one of the two ATPs is tightly bound in a nonexchangeable form. A molecular dynamics simulation of the ATP-binding process in BtuCD (an ABC protein that is the vitamin B 12 importer in Escherichia coli) favors the idea of an asymmetrical occluded state (60). This study found that, if both nucleotides were docked in identical positions relative to the Walker A and Walker B of each NBD, MgATP binding occurred across the dimer interface of one NBD.…”

Section: The Reaction Intermediate Of Pgp With Occluded Atpmentioning

confidence: 84%

About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work

Sauna

Ambudkar

2007

Molecular Cancer Therapeutics

133

100

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The efflux of drugs by the multidrug transporter Pglycoprotein (Pgp; ABCB1) is one of the principal means by which cancer cells evade chemotherapy and exhibit multidrug resistance. Mechanistic studies of Pgp-mediated transport, however, transcend the importance of this protein per se as they help us understand the transport pathway of the ATP-binding cassette proteins in general. The ATP-binding cassette proteins comprise one of the largest protein families, are central to cellular physiology, and constitute important drug targets. The functional unit of Pgp consists of two nucleotide-binding domains (NBD) and two transmembrane domains that are involved in the transport of drug substrates. Early studies postulated that conformational changes as a result of ATP hydrolysis were transmitted to the transmembrane domains bringing about drug transport. More recent structural and biochemical studies on the other hand suggested that ATP binds at the interface of the two NBDs and induces the formation of a closed dimer, and it has been hypothesized that this dimerization and subsequent ATP hydrolysis powers transport. Based on the mutational and biochemical work on Pgp and structural studies with isolated NBDs, we review proposed schemes for the catalytic cycle of ATP hydrolysis and the transport pathway. [Mol Cancer Ther 2007; 6(1):13 -23]

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“…However, it has been shown that some of these transporters can be partially uncoupled in reconstituted systems [81,82]. A quest to understand the uncoupling process has led to extensive evidence that the two NBDs bind and hydrolyze ATP in a cooperative manner [83][84][85][86].…”

Section: Putative Catalytic Mechanism Of Abc Transporters Of the "Expmentioning

confidence: 99%

Structure and mechanism of ATP-dependent phospholipid transporters

López‐Marqués

Poulsen

Bailly

et al. 2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background: ATP-binding cassette (ABC) transporters and P4-ATPases are two large and seemingly unrelated families of primary active pumps involved in moving phospholipids from one leaflet of a biological membrane to the other. Scope of review: This review aims to identify common mechanistic features in the way phospholipid flipping is carried out by two evolutionarily unrelated families of transporters. Major conclusions: Both protein families hydrolyze ATP, although they employ different mechanisms to use it, and have a comparable size with twelve transmembrane segments in the functional unit. Further, despite differences in overall architecture, both appear to operate by an alternating access mechanism and during transport they might allow access of phospholipids to the internal part of the transmembrane domain. The latter feature is obvious for ABC transporters, but phospholipids and other hydrophobic molecules have also been found embedded in P-type ATPase crystal structures. Taken together, in two diverse groups of pumps, nature appears to have evolved quite similar ways of flipping phospholipids. General significance: Our understanding of the structural basis for phospholipid flipping is still limited but it seems plausible that a general mechanism for phospholipid flipping exists in nature. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

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Conformational Transitions Induced by the Binding of MgATP to the Vitamin B12 ATP-binding Cassette (ABC) Transporter BtuCD

Cited by 88 publications

References 57 publications

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work

Structure and mechanism of ATP-dependent phospholipid transporters

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