Robert Berendes

Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.

Annexin V: the key to understanding ion selectivity and voltage regulation?

Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.

A rapid and efficient purification method for recombinant annexin V for biophysical studies

Annexin V binds in a calcium‐dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X‐ray crystallography and electron microscopy in order to understand the structure‐function relationships of the ion channel activity. We describe here a method to obtain very pure reeombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium‐mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion‐exchange chromatography and elutes as a single peak free of any detectable contaminants.

Annexin V membrane interaction : an electrostatic potential study

The possible role of electrostatic interactions for membrane binding and pore formation of annexin V has been analysed on the basis of a simple dielectric model. It is suggested that the binding of phospholipids to annexin V is regulated, at least initially, by the proteins electrostatic potential. The calculations show that a strong local gradient of the electrostatic potential exists at the membrane-protein interface and a membrane pore may be generated by electroporation. The observed specificity and regulation of ion conduction is suggested to reside in the protein part of the pore. On the basis of the three-dimensional structures of the protein and its hypothetical membrane complex, and electrophysiological measurements, a mechanical model of the transmembrane voltage regulation of the annexins ion conduction properties is proposed.

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2

A new assay for the calcium channel activity of annexin V was developed. The calcium‐sensitive fluorescence indicator, fura‐2, was incorporated into large unilamellar vesicles (LUV). After establishing a calcium gradient across the liposomal membranes, native or mutated annexin V was added. The resulting calcium influx into the LUV detected through the fluorescence changes of fura‐2 was used as a qualitative test for the electrophysiological properties of annexin V.

Annexin V: Structure-function analysis of a voltage-gated, calcium-selective ion channel

The structural basis of ion channel function remained mostly a mystery for more than 140 years since the existence of water-filled pores through membranes was first postulated (Brucke, 1843). Despite intense efforts in the determination of the electrophysiological properties of ion channels (for a review see Hille, 1992), starting with the seminal work of Hodgkin and Huxley, the field still suffers from the lack of high-resolution structures of ion channel proteins. The first determination of the structure of a membrane protein, the photosynthetic reaction center (Deisenhofer et al., 1985), was a flash of hope for the crystallization of membrane-integrated ion channel proteins in order to understand their selectivity, conductance regulation, and voltage-gating. But still, no crystals of ion channel proteins diffracting x-rays to high resolution could be obtained. Therefore, channel model systems with known crystal structures, such as the porins (e.g., Weiss et al., 1991) or colicins (e.g., Parker et al., 1989), were valuable. Unfortunately, these proteins do not combine the main features of typical ion channels mentioned above. To compensate for this shortcoming extensive investigations by mutational analysis (see Catterall, 1988 or Miller, 1991 for reviews) and molecular modeling (see Guy and Seetharamulu, 1992; Durell and Guy, 1992) of ion channels and of pore-forming peptides (see Sansom, 1991 for a review) were carried out. In 1990, annexin V was structurally characterized as the first ion channel protein, thus paving the way, in combination with electrophysiological analysis of annexin V mutants, for an understanding of ion channel function in molecular terms.