Primary events in the colicin translocon: FRET analysis of colicin unfolding initiated by binding to BtuB and OmpF.
Cellular import of colicin E3 is initiated by high affinity binding of the colicin receptor-binding (R) domain to the vitamin B(12) (BtuB) receptor in the Escherichia coli outer membrane. The BtuB binding site, at the apex of its extended coiled-coil R-domain, is distant from the C-terminal nuclease domain that must be imported for expression of cytotoxicity. Based on genetic analysis and previously determined crystal structures of the R-domain bound to BtuB, and of an N-terminal disordered segment of the translocation (T) domain inserted into the OmpF porin, a translocon model for colicin import has been inferred. Implicit in the model is the requirement for unfolding of the colicin segments inserted into OmpF. FRET analysis was employed to study colicin unfolding upon interaction with BtuB and OmpF. A novel method of Cys-specific dual labeling of a native polypeptide, which allows precise placement of donor and acceptor fluorescent dyes on the same polypeptide chain, was developed. A decrease in FRET efficiency between the translocation and cytotoxic domains of the colicin E3 was observed upon colicin binding in vitro to BtuB or OmpF. The two events were independent and additive. The colicin interactions with BtuB and OmpF have a major electrostatic component. The R-domain Arg399 is responsible for electrostatic interaction with BtuB. It is concluded that free energy for colicin unfolding is provided by binding of the R- domain to BtuB and binding/insertion of the T-domain to/into OmpF.
Zakharov
Stanislav D
SD
Department of Biological Sciences and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907-2054, USA.
Sharma
Onkar
O
Zhalnina
Mariya V
MV
Cramer
William A
WA
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003087:Colicins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D018272:Porins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021381:Protein Transport; D013194:Staining and Labeling; D055672:Static Electricity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; C109237:OmpF protein; D018272:Porins; D013438:Sulfhydryl Compounds; D003545:Cysteine