The extracellular gate shapes the energy profile of an ABC exporter

Abstract

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...

Nanobodies—Useful Tools for Allergy Treatment?

“…Non-immune libraries are typically much larger than immune libraries and a ribosome display was suggested for the initial selection round of such large libraries to work with higher concentrations of nanobody variants than in case of phage display ( 79 , 80 ). Synthetic libraries combined with different selection procedures were successfully used to obtain conformationally selective nanobodies against G protein-coupled receptors ( 78 ), sybodies against very challenging targets such as the heterodimeric bacterial ABC exporterTM287/288 ( 81 ) or the intracellular KDEL receptor ( 82 ) to name a few examples from many others.…”

Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters

“…1c). Separation of helices at the TMD apex to open the extracellular gate requires the release of contacts stabilizing the inward-facing state 31 , which, as explained below, is relevant to substrate-stimulated conformational cycling 32 .…”

Structures of ABC transporters: handle with care

“…According to the first model (which constitutes the prevailing mechanism), binding of two ATP molecules leads to the formation of the closed NBD sandwich dimer and results in a conformational change at the TMDs from the IF to the OF state, which is sufficient to translocate one substrate molecule across the membrane [15,[56][57][58][59]. ATP hydrolysis is then required to initiate the dissociation of the closed NBD dimer, in order to reset the transporter back to its IF state and keep the transport cycle going [60,61].…”

“…in case of TM287/288 [15], BmrCD [62]; TmrAB [87] and ABCB1 [16]), whereas for MsbA [69] it fully switches the transporter to the OF state. The inability of nonhydrolyzable ATP analogues to reliably stabilize the closed NBD dimer was frequently overcome by mutating the catalytic Walker B glutamate residue to glutamine, alanine, or glycine and thereby trap the Occ or OF state in the presence of ATP [52,56,[59][60][61]67,88]. Cases in which the Walker B glutamate mutation trick did not work to obtain OF or outward-occluded structures have not been reported.…”