Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

Benesh Joseph,Gunnar Jeschke,Birke A Goetz,Kaspar P Locher,Enrica Bordignon

doi:10.1074/jbc.m111.269472

Abstract

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

Highlights

“two-state, alternating access” mechanistic model for both ATP-binding cassette (ABC) exporters and importers [4]
For type I ABC importers the details of the alternating access mechanism have been confirmed by a large body of experimental evidence, including the crystal structures showing the maltose transporter in different states during the nucleotide cycle [5,6,7,8]
The present study reveals how the transmembrane gates in the vitamin B12 transporter BtuCD-F respond to the presence of BtuF, substrate, and nucleotides

Summary

Introduction

“two-state, alternating access” mechanistic model for both ABC exporters and importers [4]. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. BtuCD-F was proven by EPR to mainly expose the NBDs to the outside of the liposomes: for all positions investigated an almost complete change of the distance distributions (without residual apo-state distances) was induced upon ATP addition (Fig. 2).

Results

Conclusion