ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.DOI: http://dx.doi.org/10.7554/eLife.20236.001

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 result in a comparable equilibrium shift and strongly reduce 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.

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288. Extensive multimicrosecond simulations in an explicit membrane/water environment show how in response to ATP binding, TM287/288 undergoes spontaneous conformational transitions from the inward-facing (IF) state via an occluded (Occ) intermediate to an outward-facing (OF) state. The latter two states have thus far not been characterized at atomic level. ATP-induced tightening of the NBD dimer involves closing and reorientation of the two NBD monomers concomitant with a closure of the intracellular TMD gate, which leads to the occluded state. Subsequently, opening at the extracellular TMD gate yields the OF conformer. The obtained mechanism imposes NBD-TMD coupling via a tight orchestration of conformational transitions, between both the two domains and also within the TMDs, ensuring that the cytoplasmic and periplasmic gate regions are never open simultaneously.

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...