Structural and Functional Fingerprint of the Mitochondrial ATP-binding Cassette Transporter Mdl1 from Saccharomyces cerevisiae

Hofacker, Matthias; Gompf, Simone; Zutz, Ariane; Presenti, Chiara; Haase, Winfried; Does, Chris van der; Model, Kirstin; Tampé, Robert

doi:10.1074/jbc.m609899200

Cited by 27 publications

(24 citation statements)

References 65 publications

(47 reference statements)

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“…Determination of the Michaelis-Menten kinetics revealed a K m,MgATP value of 0.9 mM, which is in good agreement to other ABC proteins (e.g. the homodimeric mitochondrial ABC transport complex MDL1 from Saccharomyces cerevisiae (29)). Comparable with other multidrug resistance proteins like LmrCD or BmrA (45,47), TmrAB exhibits a high basal ATPase activity of 9 s Ϫ1 .…”

Section: Discussionsupporting

confidence: 75%

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus

Zutz¹,

Hoffmann²,

Hellmich

et al. 2011

Journal of Biological Chemistry

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ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ϳ46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a K m of 0.9 mM and k cat of 9 s ؊1 at 68°C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.

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Section: Discussionsupporting

confidence: 75%

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus

Zutz¹,

Hoffmann²,

Hellmich

et al. 2011

Journal of Biological Chemistry

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“…1 B and C). The open-apo conformation of other ABC transporters has also been observed in structural studies of BmrA (YvcC) (57) and Mdl1 (58), where the NBDs are apart. Further, the disassociation of NBDs is also consistent with the x-ray structures of several isolated ABC domains that do not form the ATP sandwich dimer in the absence of nucleotide (reviewed in ref.…”

Section: Discussionmentioning

confidence: 67%

Flexibility in the ABC transporter MsbA: Alternating access with a twist

Ward¹,

Reyes

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

724

1,007

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“…Notably, and in contrast to other ABC systems (14,35,36), Pdr5 is the first ABC transporter for which a mutation of the H-loop histidine to alanine did not affect the steady-state ATPase activity. Strikingly, the observation that the H1068A mutation abolishes R6G transport without any impact on the observed ATPase activity clearly represents a novelty in the field of ABC transporters (Fig.…”

Section: Discussionmentioning

confidence: 67%

A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

Ernst

Kueppers

Klein

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

137

219

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The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed ''catalytic carboxylate'' (E1036) and the ''catalytic dyad histidine'' (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.ATPase activity ͉ multidrug resistance ͉ substrate recognition

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Structural and Functional Fingerprint of the Mitochondrial ATP-binding Cassette Transporter Mdl1 from Saccharomyces cerevisiae

Cited by 27 publications

References 65 publications

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus

Flexibility in the ABC transporter MsbA: Alternating access with a twist

A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5

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