Essential Role of B-helix Calcium Binding Sites in Annexin V-Membrane Binding

Abstract

Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule. In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total). We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (<1% saturation of available membrane binding sites). Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites. The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity. Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent. The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations. This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity.The annexins are a family of calcium-dependent membranebinding proteins with diverse functions (1-4). Annexin V is a typical member of this protein family, which is becoming important as a means to measure cell death in vivo in cancer chemotherapy, organ transplant rejection, and myocardial infarction (5-10), creating a need to better understand structural and functional properties relevant to its clinical use. Novel engineered forms of annexin V are being developed to improve its utility for imaging (11,12), and there has been a recent report of a single-domain version of annexin V that may be useful for imaging cell death in vivo (13).Calcium binding sites in annexins are of three structural types: AB (or Type II), DE (or Type III), and B or ABЈ (14). In these binding sites, calcium ligands consist of one to three carbonyl oxygens from the protein backbone, and one carboxyl group from an Asp or Glu residue; sites are often named based on the domain (1 through 4) and ␣-helices (A through E), which contribute the backbone carbonyl ligands. However, the number of calcium ions observed in different structures varies widely; for example, anywhere from 2 or 3 (15, 16) to 10 (17) calcium ions have been observed in different crystal forms of annexin V. Thus, it has been difficult to determine which calcium binding sites are functionally important based on structural data alone. Previous work with annexin V mutants indicated that the AB site in domain 1 was the most impor...

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

“…Materials-Recombinant proteins were prepared as described: human wild-type annexin V (26) and annexin V-128 (23). Both proteins were quantitated by absorbance at 280 nm using an extinction coefficient of 21,500 M Ϫ1 cm Ϫ1 (27).…”

“…Construction, Expression, and Fluorescent Labeling of Annexin V Mutants-Expression vectors were constructed from the base plasmid pJ128 (23), which expresses a 325-amino acid protein (named annexin V-128) similar to wild-type human annexin V but with these changes: 1) an N-terminal extension of (Met)-Ala-Gly-Gly-Cys-Gly-His; 2) deletion of the initiator Met at position 1 of wild-type annexin V; 3) a point mutation C316S. Point mutations were made with the QuikChange Multisitedirected mutagenesis kit (Stratagene, La Jolla, CA).…”

“…Each annexin V mutant protein was named with an arbitrary numerical suffix. For protein expression, plasmids were transformed into Escherichia coli strain Tuner (DE3) pLacI (Novagen, Madison, WI) and expressed and purified as described (23). The yield and chromatographic behavior of all mutant proteins were comparable with that of annexin V-128, suggesting that all proteins were stable and did not have grossly altered conformations.…”

“…Recently, we developed methods for quantitative measurement of the binding of annexin V to cells and phospholipid vesicles, which allow one to determine the free energy of binding for individual protein molecules (22,23). These methods rely on calcium titration performed at low levels of membrane occupancy, which overcomes the difficulties of analyzing a full binding isotherm previously identified by Bazzi and Nelsestuen (24,25).…”

“…These methods rely on calcium titration performed at low levels of membrane occupancy, which overcomes the difficulties of analyzing a full binding isotherm previously identified by Bazzi and Nelsestuen (24,25). This method allowed us to determine that under physiological conditions there are about eight high-affinity calcium binding sites, formed by the A and B helices in each of the four domains, that account for about 70% of the free energy of binding (23). In this study, we sought to investigate the source of the remaining ϳ30% of binding energy, and thereby gain insight into the structural and mechanistic basis for the binding reac-tion.…”