Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter

Göddeke, Hendrik; Timachi, M. Hadi; Hutter, Cedric A. J.; Galazzo, Laura; Seeger, Markus A.; Karttunen, Mikko; Bordignon, Enrica; Schäfer, Lars V.

doi:10.1021/jacs.7b12944

Cited by 40 publications

(58 citation statements)

References 72 publications

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“…The ATP-bound OF state with completely closed NBDs has been referred to as the high-energy state of the transport cycle and in some instances it was proposed that ATP hydrolysis is needed to populate the OF state at all 10,25 . In contrast, we and others have previously stipulated that ATP binding alone is sufficient for IF-OF conversion and substrate release 9,11,26 , in agreement with the ATP-switch model 27 . It should be noted that while the opened extracellular gate indeed represents a high-energy state, the opposite is in fact true for the closed NBD dimer.…”

Section: Discussionsupporting

confidence: 76%

“…With a RMSD of 1.73 Å, the structure of TM287/288 most closely resembles the structure of Sav1866 and is similar to the OF state of TM287/288 predicted by MD simulations (Fig. S5) 11 . Also the degree of NBD closure and extracellular gate opening is highly similar between TM287/288 and Sav1866 (Fig.…”

Section: Transition From If To Of State Renders Tm287/288 More Symmetricsupporting

confidence: 68%

“…A sybody stabilizing the opened extracellular wing or mutations weakening the closed extracellular gate both shifted the conformational equilibrium towards the OF state. Previous experimental and computational studies have uncovered that NBD closure precedes extracellular gate opening during the IF-OF transition 11,24 , and DEER analyses have revealed that extracellular gate opening can be partial while NBD closure is complete 9, 10, 25 . Hence, there seems to be an inbuilt mechanical principle that the extracellular gate is energetically costly to open.…”

Section: Discussionmentioning

confidence: 99%

“…To probe the atomic details of the conformational dynamics underlying the IF-OF transition, we performed MD simulations of TM287/288 in a lipid bilayer starting from the IF crystal structure (PDB: 4Q4A), after docking a second ATP-Mg molecule into the consensus site 11 and introducing the 2xDtoA mutations in the extracellular gate. As in our previous MD simulations of wildtype TM287/288 11 , we observed spontaneous large-scale conformational transitions from the IF conformation via an Occ state to an OF conformation; this complete transition was observed in 3 out of 20 independent 500 ns simulations ( Fig.…”

Section: Conserved Aspartates Seal the Extracellular Gatementioning

confidence: 99%

“…Similar observations were reported for ABCB1 10 . Unbiased MD simulations of TM287/288 uncovered spontaneous conformational transitions from the IF state via an Occ intermediate to the OF state 11 . Many simulations remained trapped in the Occ state, suggesting that extracellular gate opening represents a major energetic barrier in the conformational cycle.…”

Section: Introductionmentioning

confidence: 99%

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The extracellular gate shapes the energy profile of an ABC exporter

Hutter

Timachi

Hürlimann

et al. 2018

Preprint

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ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state. RESULTS Conformational trapping of TM287/288Having solved two closely related IF structures of TM287/288, our aim was to obtain an atomic structure of this heterodimeric ABC exporter in its OF state. DEER analyses revealed that TM287/288 carrying the TM288 E517Q mutation in the Walker B motif of the consensus site (EtoQ mutation) was almost completely trapped in the OF state in the presence of ATP-Mg and ATPγS-Mg 9 . To further decrease the residual ATPase activity of the EtoQ mutant (turnover of 0.02 min -1 ) by a factor of 6.5, we instead introduced the EtoA mutation. In addition, we generated single domain antibodies (nanobodies) that exclusively recognize the OF state of TM287/288. To this end, alpacas were immunized with outward-facing TM287/288 containing a cross-linked tetrahelix bundle motif 13 (see Materials and Methods). This approach yielded nanobody Nb_TM#1 binding exclusively to TM287/288 in the presence (but not in the absence) of ATP, as shown by surface plasmon resonance (SPR) (Fig. 1d). However, crystals obtained with Nb_TM#1 did not diffract well enough to build a reliable model. Therefore, we selected synthetic nanobodies (sybodies) against TM287/288(EtoA) in the presence of ATP-Mg completely in vitro 14 . Thereby, more than ten OF-specific sybodies were generated and sybody Sb_TM#35 was successfully used to solve the OF structure of TM287/288(EtoA) in the presence of ATPγS-Mg at 3.2 Å resolution ( Fig. 1a, Table S1). Structure of OF TM287/288 with a sybody bound to an extracellular wingSybody Sb_TM#35 binds on top of an extracellular wing of TM287/288 ( Fig. 1a) and was crucially involved in establishing crystal contacts (Fig. S1). Binding is mediated by aromatic residues of all three complementary determining regions (CDRs) of the sybody, which are wedged between transmembrane helices (TMs) 1 and 2 of TM287 and TMs 5' and 6' of TM288 ( Fig. 2a). Since Sb_TM#35 only binds in the presence of ATP (Fig. 1d), we hypothesized that it interferes with the catalytic cycle o...

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Section: Discussionsupporting

confidence: 76%

Section: Transition From If To Of State Renders Tm287/288 More Symmetricsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Conserved Aspartates Seal the Extracellular Gatementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The extracellular gate shapes the energy profile of an ABC exporter

Hutter

Timachi

Hürlimann

et al. 2018

Preprint

Self Cite

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Structures of ABC transporters: handle with care

2020

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In the past two decades, the ATP-binding cassette (ABC) transporters' field has undergone a structural revolution. The importance of structural biology to the development of the field of ABC transporters cannot be overstated, as the ensemble of structures not only revealed the architecture of ABC transporters but also shaped our mechanistic view of these remarkable molecular machines. Nevertheless, we advocate that the mechanistic interpretation of the structures is not trivial and should be carried out with prudence. Herein, we bring several examples of structures of ABC transporters that merit re-interpretation via careful comparison to experimental data. We propose that it is of the upmost importance to place new structures within the context of the available experimental data.

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The role of the degenerate nucleotide binding site in type I ABC exporters

2020

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ATP-binding cassette (ABC) transporters are fascinating molecular machines that are capable of transporting a large variety of chemically diverse compounds. The energy required for translocation is derived from binding and hydrolysis of ATP. All ABC transporters share a basic architecture and are composed of two transmembrane domains and two nucleotide binding domains (NBDs). The latter harbor all conserved sequence motifs that hallmark the ABC transporter superfamily. The NBDs form the nucleotide binding sites (NBSs) in their interface. Transporters with two active NBSs are called canonical transporters, while ABC exporters from eukaryotic organisms, including humans, frequently have a degenerate NBS1 containing noncanonical residues that strongly impair ATP hydrolysis. Here, we summarize current knowledge on degenerate ABC transporters. By integrating structural information with biophysical and biochemical evidence of asymmetric function, we develop a model for the transport cycle of degenerate ABC transporters. We will elaborate on the unclear functional advantages of a degenerate NBS.

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Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter

Cited by 40 publications

References 72 publications

The extracellular gate shapes the energy profile of an ABC exporter

The extracellular gate shapes the energy profile of an ABC exporter

Structures of ABC transporters: handle with care

The role of the degenerate nucleotide binding site in type I ABC exporters

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