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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 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.
Highlights
ABC exporters harness the energy of ATP to pump substrates across membranes
Type I exporters are the best studied class of ABC exporters and minimally consist of two transmembrane domains (TMDs) each comprising six transmembrane helices and two nucleotide binding domains (NBDs) that are universally conserved among all ABC transporters
Two closely related IF structures of TM287/288 were solved by X-ray crystallography either containing one AMP-PNP molecule bound to the degenerate site or no nucleotide
Summary
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. 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. 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. In contrast to most other IF structures of ABC exporters, the opened NBDs of TM287/288 are only partially separated due to contacts mediated by the degenerate site D-loop, whereas the consensus site D-loop was found to allosterically couple ATP binding at the degenerate site to ATP hydrolysis at the consensus site[8].
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