Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes.
Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb(3)). Tryptophan residues of STxB, located at the protein-membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb(3)-containing vesicles suggest a complex STxB/Gb(3) docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein-membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.
Pina
David G
DG
Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande C8, 1749-016 Lisboa, Portugal. dgp28@cam.ac.uk
Johannes
Ludger
L
Castanho
Miguel A R B
MA
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002021:Buffers; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005915:Globosides / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D022621:Shiga Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014281:Trihexosylceramides / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D002021:Buffers; D005915:Globosides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011994:Recombinant Proteins; D014281:Trihexosylceramides; D053835:Unilamellar Liposomes; D014867:Water; D015060:1,2-Dipalmitoylphosphatidylcholine; C018549:globotriaosylceramide; D022621:Shiga Toxin; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D014364:Tryptophan; C028694:1-palmitoyl-2-oleoylphosphatidylcholine