Single-molecule visualization of conformational changes and substrate transport in the vitamin B12 ABC importer BtuCD-F

Joris M H Goudsmits,Dirk Jan Slotboom,Antoine M Van Oijen

doi:10.1038/s41467-017-01815-7

Joris M H Goudsmits, Dirk Jan Slotboom + Show 1 more

Open Access

https://doi.org/10.1038/s41467-017-01815-7

Copy DOI

Abstract

ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Binding and hydrolysis of ATP by their highly conserved nucleotide-binding domains drive conformational changes of the complex that mediate transport of substrate across the membrane. The vitamin B12 importer BtuCD-F in Escherichia coli is an extensively studied model system. The periplasmic soluble binding protein BtuF binds the ligand; the transmembrane and ATPase domains BtuCD mediate translocation. Here we report the direct observation at the single-molecule level of ATP, vitamin B12 and BtuF-induced events in the transporter complex embedded in liposomes. Single-molecule fluorescence imaging techniques reveal that membrane-embedded BtuCD forms a stable complex with BtuF, regardless of the presence of ATP and vitamin B12. We observe that a vitamin B12 molecule remains bound to the complex for tens of seconds, during which several ATP hydrolysis cycles can take place, before it is being transported across the membrane.

Highlights

ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins
Extensive structural and biochemical characterisation of BtuCD alone or in complex with BtuF has provided the framework for understanding the mechanism of vitamin B12 transport: crystal structures have revealed several intermediate states in the transport cycle[14,15,16], the gating mechanism of the transmembrane domains (TMDs) upon ATP hydrolysis has been investigated with EPR techniques[17,18], and biochemical characterisation of the complex in detergent and proteoliposomes has given insight into the molecular steps underlying transport[19,20]
For each condition in d–f around 1000 single-molecule fluorescence traces were analysed space20? Is the substrate immediately transported, and is ATP merely required to reset the transporter20? Why is the ATPase activity at least one order of magnitude higher compared to transport of vitamin B12—in other words, why is there no strong coupling between ATP hydrolysis and substrate translocation such as observed for the well-studied type I maltose importer MalFGK21? In order to address these questions, we have employed single-molecule fluorescence techniques to follow individual BtuCD-F proteins reconstituted in liposomes through time and to directly observe steps of the transport cycle

Summary

Introduction

ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Extensive structural and biochemical characterisation of BtuCD alone or in complex with BtuF has provided the framework for understanding the mechanism of vitamin B12 transport: crystal structures have revealed several intermediate states in the transport cycle[14,15,16], the gating mechanism of the TMDs upon ATP hydrolysis has been investigated with EPR techniques[17,18], and biochemical characterisation of the complex in detergent and proteoliposomes has given insight into the molecular steps underlying transport[19,20] These studies all suggest that BtuCD-F employs a different mechanism than that described by the alternating-access model for type I importers. We interpret our results in the light of different existing models for transport by BtuCD-F

Methods

Results

Conclusion