Probing receptor-translocator interactions in the oligopeptide ABC transporter by fluorescence correlation spectroscopy.
The oligopeptide transporter Opp is a five-component ABC uptake system. The extracytoplasmic lipid-anchored substrate-binding protein (or receptor) OppA delivers peptides to an integral membrane complex OppBCDF (or translocator), where, on ATP binding and hydrolysis, translocation across the membrane takes place. OppA and OppBCDF were labeled with fluorescent probes, reconstituted into giant unilamellar vesicles, and the receptor-translocator interactions were investigated by fluorescence correlation spectroscopy. Lateral mobility of OppA was reduced on incorporation of OppBCDF into giant unilamellar vesicles, and decreased even further on the addition of peptide. Fluorescence cross-correlation measurements revealed that OppBCDF distinguished liganded from unliganded OppA, binding only the former. Addition of ATP or its nonhydrolyzable analog AMP-PNP resulted in release of OppA from OppBCDF. In vanadate-trapped "transition state" conditions, OppA also was not bound by OppBCDF. A model is presented in which ATP-binding to OppDF results in donation of peptide to OppBC and simultaneous release of OppA. ATP-hydrolysis would complete the peptide translocation and reset the transporter for another catalytic cycle. Implications in terms of a general transport mechanism for ABC importers and exporters are discussed.
Doeven
Mark K
MK
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
van den Bogaart
Geert
G
Krasnikov
Victor
V
Poolman
Bert
B
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D008074:Lipoproteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D015336:Molecular Probe Techniques; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D053835:Unilamellar Liposomes / Q000737:chemistry*
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D053835:Unilamellar Liposomes; C046506:oligopeptide-binding protein, bacteria; C072861:OppB protein, Salmonella