Synergy between Conserved ABC Signature Ser Residues in P-glycoprotein Catalysis

Tombline, Gregory; Bartholomew, Lori A.; Gimi, Khursheed; Tyndall, Grace A.; Senior, Alan E.

doi:10.1074/jbc.m311964200

Cited by 57 publications

(56 citation statements)

References 45 publications

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“…Similar findings were most recently observed for P-glycoprotein (36). Mutation of the serine in one of the C-loops of Pglycoprotein to alanine in each NBD reduces ATPase activity to 66%, and the double mutant possesses 3% of the ATPase activity of wild type P-glycoprotein.…”

Section: Discussionsupporting

confidence: 69%

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

Chen

Abele

Tampé

2004

Journal of Biological Chemistry

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The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.

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Section: Discussionsupporting

confidence: 69%

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

Chen

Abele

Tampé

2004

Journal of Biological Chemistry

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“…Sensitive assays were therefore conducted using radioactive [␥-32 P]ATP as described previously (31). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Mutations at positions 552 and 1197 of mouse MDR3 Pgp were generated by PCR mutagenesis of pAlt-mdr3N and pAlt-mdr3.6C, respectively, as described previously (31). The following mutant species were generated: E552Q/E1197Q, E552A/E1197A, E552D/E1197D, and E552K/E1197K.…”

Section: Construction Of Mutant Pgp; Expression Of Mutant Pgp In Pichmentioning

confidence: 99%

“…The Pgp concentration was determined as described previously (31). Briefly, increasing amounts of mutant Pgp (0.2-2.0 g) were subjected to SDS-gel electrophoresis on 10% gels alongside a similar set of samples of a reference pure wild-type preparation whose concentration had previously been determined accurately by amino acid analysis.…”

Section: Purification and Quantitation Of Pgpmentioning

confidence: 99%

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Combined Mutation of Catalytic Glutamate Residues in the Two Nucleotide Binding Domains of P-glycoprotein Generates a Conformation That Binds ATP and ADP Tightly

Tombline

Bartholomew

Urbatsch

et al. 2004

Journal of Biological Chemistry

Self Cite

125

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Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/ E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/ E1197A bound MgATP and MgADP (1 mol/mol) with K d 9.2 and 92 M, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.P-glycoprotein (Pgp) 1 is a plasma membrane protein that confers multidrug resistance by virtue of its ability to exclude hydrophobic compounds from cells in an ATP-dependent fashion. Anticancer drugs and AIDS protease inhibitors are among the numerous compounds transported by Pgp, and there is the realization that a substantial number of future new drug candidates will similarly be transport substrates, so much current interest centers on strategies aimed at disabling or circumventing Pgp. Consisting of two transmembrane domains and two nucleotide binding domains (NBDs), Pgp displays the typical architecture of the ABC transporter family of membrane transporters. There is as yet no reported high resolution structure of Pgp, but structures of several homologous ABC transporters, and of isolated NBDs, have been published recently. For recent reviews of Pgp structure and function see Refs. 1-6.Our laboratory has focused on the mechanism of ATP hydrolysis and ATP hydrolysis-driven transport. Procedures for large scale preparation of pure human MDR1 (7) and mouse MDR3 2 protein (8) have recently facilitated such studies. Earlier work had established that Pgp showed drug-stimulated ATPase activity (9, 10) and that hydrolysis of ATP occurred in both NBDs (11). It was clear from studies with inhibitors N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (10, 12-14), mutations in the catalytic sites (15, 16), and vanadate-trapping experiments (17) that the two NBDs cooperated strongly and mandatorily for hydrolysis, and an alternating sites mechanism was proposed in 1995 (18). This mechanism envi...

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“…For CFTR, there is indirect evidence that ATP must bind to both sites to open the channel [59,60]. Finally, co-operative ATP binding suggests a role for two ATP molecules [50,[61][62][63][64]. However, it is possible to envisage an ABC transporter where one ATP is sufficient to stabilise the 'closed dimer' depending on the nature of the dimer interface.…”

Section: Stoichiometry Of Atp Binding and Hydrolysismentioning

confidence: 99%

Structure and function of ABC transporters: the ATP switch provides flexible control

Linton

Higgins

2006

Pflugers Arch - Eur J Physiol

190

160

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ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that facilitate the transbilayer movement of ligands. They comprise, minimally, two transmembrane domains, which impart ligand specificity, and two nucleotide-binding domains (NBDs), which power the transport cycle. Almost 25 years of biochemistry is reviewed in light of the recent structure analyses resulting in the ATP-switch model for function in which the NBDs switch between a dimeric conformation, closed around two molecules of ATP, and a nucleotide-free, dimeric 'open' conformation. The flexibility of this switching mechanism has evolved to provide different kinetic control for different transporters and has also been co-opted to diverse functions other than transmembrane transport.

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Synergy between Conserved ABC Signature Ser Residues in P-glycoprotein Catalysis

Cited by 57 publications

References 45 publications

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

Combined Mutation of Catalytic Glutamate Residues in the Two Nucleotide Binding Domains of P-glycoprotein Generates a Conformation That Binds ATP and ADP Tightly

Structure and function of ABC transporters: the ATP switch provides flexible control

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