title: GTP-induced membrane binding and ion channel activity of annexin VI: is annexin VI a GTP biosensor?
abstract: Annexin VI (AnxVI) formed ion channels in planar lipid bilayers that were induced by the addition of millimolar guanosine 5'-triphosphate (GTP) at pH 7.4 and that were not accompanied by a penetration of the protein into the membrane hydrophobic region. GTP-influenced interactions of AnxVI with Ca2+/liposomes produced small structural alterations as revealed by circular dichroism and infrared spectroscopies. Guanosine 5'-3-O-(thio)-triphosphate (GTPgammaS) binding to AnxVI, promoted by the photorelease of GTPgammaS from GTPgammaS[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged-GTPgammaS), affected three to four amino acid residues of AnxVI in the presence of Ca2+/liposomes, while about eight or nine amino acid residues were altered in their absence. This suggested that the nucleotide-binding site overlapped the lipid-binding domain of AnxVI. The binding of the fluorescent GTP analog, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP) to AnxVI was optimal in the presence of Ca2+/liposomes, with a dissociation constant (K(d)) of 1 microM and stoichiometry of 1. TNP-GTP promoted fluorescence resonance energy transfer from tryptophan residues to the nucleotide. Ion conductance and fluorescence measurements of the C- and N-terminal fragments of AnxVI indicated distinct GTP-binding properties, suggesting that the existence of the GTP-induced ion channel activity of AnxVI is associated with the flexibility of the two halves of the protein. Such structural flexibility could contribute to a molecular mechanism of AnxVI acting as a GTP biosensor.
authors.affiliation: Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Kirilenko
authors.forename: Marcin
authors.initials: M
authors.lastname: Golczak
authors.forename: Slawomir
authors.initials: S
authors.lastname: Pikula
authors.forename: Rene
authors.initials: R
authors.lastname: Buchet
authors.forename: Joanna
authors.initials: J
authors.lastname: Bandorowicz-Pikula
mesh_terms: D017317:Annexin A6 / Q000302:isolation & purification / Q000378:metabolism*; D015374:Biosensing Techniques; D002942:Circular Dichroism; D003902:Detergents; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics*; D006160:Guanosine Triphosphate / Q000502:physiology*; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology*; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013055:Spectrophotometry, Infrared
chemical_list: D017317:Annexin A6; D003902:Detergents; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D017830:Octoxynol; C010615:Nonidet P-40; D019204:GTP-Binding Proteins