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• GTP-Induced Membrane Binding and Ion Channel Activity of Ann...

  GTP-Induced Membrane Binding and Ion Channel Activity of Annexin VI: Is Annexin VI a GTP Biosensor? Biophysical Journal, Volume 82, Issue 5, 1 May 2002, Pages 2737-2745 Aneta Kirilenko, Marcin Golczak, Slawomir Pikula, Rene Buchet and Joanna Bandorowicz-Pikula 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 Ca/liposomes produced small structural alterations as revealed by circular dichroism and infrared spectroscopies. Guanosine 5′-3--(thio)-triphosphate (GTPS) binding to AnxVI, promoted by the photorelease of GTPS from GTPS[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged-GTPS), affected three to four amino acid residues of AnxVI in the presence of Ca/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′)--(2,4,6-trinitrophenyl)guanosine 5′-triphosphate (TNP-GTP) to AnxVI was optimal in the presence of Ca/liposomes, with a dissociation constant () of 1M 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. Abstract | Full Text | PDF (148 kb)

• Structure of Human Annexin A6 at the Air-Water Interface and...

  Structure of Human Annexin A6 at the Air-Water Interface and in a Membrane-Bound State Biophysical Journal, Volume 87, Issue 2, 1 August 2004, Pages 1215-1226 Marcin Golczak, Aneta Kirilenko, Joanna Bandorowicz-Pikula, Bernard Desbat and Slawomir Pikula Abstract We postulate the existence of a pH-sensitive domain in annexin A6 (AnxA6), on the basis of our observation of pH-dependent conformational and orientation changes of this protein and its N- (AnxA6a) and C-terminal (AnxA6b) halves in the presence of lipids. Brewster angle microscopy shows that AnxA6, AnxA6a, and AnxA6b in the absence of lipids accumulate at the air-water interface and form a stable, homogeneous layer at pH below 6.0. Under these conditions polarization modulation IR absorption spectroscopy reveals significant conformational changes of AnxA6a whereas AnxA6b preserves its -helical structure. The orientation of protein -helices is parallel with respect to the interface. In the presence of lipids, polarization modulation IR reflection absorption spectroscopy experiments suggest that AnxA6a incorporates into the lipid/air interface, whereas AnxA6b is adsorbed under the lipid monolayer. In this case AnxA6a regains its -helical structures. At a higher pressure of the lipid monolayer the average orientation of the -helices of AnxA6a changes from flat to tilted by 45° with respect to normal to the membrane interface. For AnxA6b no such changes are detected, even at a high pressure of the lipid monolayer—suggesting that the putative pH-sensitive domain of AnxA6 is localized in the N-terminal half of the protein. Abstract | Full Text | PDF (415 kb)

• Membrane-Induced Folding and Structure of Membrane-Bound Ann...

  Membrane-Induced Folding and Structure of Membrane-Bound Annexin A1 N-Terminal Peptides: Implications for Annexin-Induced Membrane Aggregation Biophysical Journal, Volume 94, Issue 5, 1 March 2008, Pages 1773-1781 Nien-Jen Hu, Jeremy Bradshaw, Hans Lauter, Julia Buckingham, Egle Solito and Andreas Hofmann Abstract Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an -helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an -helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into -helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor. Abstract | Full Text | PDF (561 kb)

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Abstract

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.