Rat Annexin V Crystal Structure: Ca ²⁺ -Induced Conformational Changes

Abstract

Annexins are a family of calcium- and phospholipid-binding proteins implicated in mediating membrane-related processes such as secretion, signal transduction, and ion channel activity. The crystal structure of rat annexin V was solved to 1.9 angstrom resolution by multiple isomorphous replacement. Unlike previously solved annexin V structures, all four domains bound calcium in this structure. Calcium binding in the third domain induced a large relocation of the calcium-binding loop regions, exposing the single tryptophan residue to the solvent. These alterations in annexin V suggest a role for domain 3 in calcium-triggered interaction with phospholipid membranes.

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

“…We also evaluated the role of Trp-187, which has been previously postulated to participate in hydrophobic interactions during membrane binding (15,16). Results showed a small but consistent decrease in binding affinity for the W187A mutant (Table S1), consistent with earlier data (33).…”

“…7C). Similarly, Trp-187 is at another protein-protein interface in the crystal structure (16). The W187A mutant also showed no change in its curve of FRET as a function of occupancy (Fig.…”

“…Thus, contributions from many hydrophobic residues would be required to explain the observed effect, no single amino acid could explain such a large difference. For example, Trp-187 has been implicated in binding to membranes (15,16), but this residue alone could account for only about 10% of the observed effect based on a predicted value of 1.8 kcal/mol for interfacial hydrophobicity. Our experimental data confirm the modest contribution of Trp-187 (see "Results").…”

“…General electrostatic interactions may occur between positively charged amino acids and negatively charged groups on the membrane. There may be insertion of specific hydrophobic residues, such as Trp-187, into the hydrophobic region of the membrane (15,16). There may also be specific interactions between protein side chains and non-phosphate components of the phospholipid head groups (14,17).…”

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

“…Similarly, proteins specific to the anionic phospholipid phosphatidylserine (PS) 2 may coordinate Ca 2ϩ ions for PS binding using conserved loop motifs as observed in annexin V (5) or directly bind PS via C2 domains as in coagulation factor V (6) and lactadherin (7) where polar side chains allow specific recognition of PS. The affinity of the proteins for the membrane may be further modulated by insertion of hydrophobic and aromatic amino acids into the bilayer (5,6,8) and/or multivalent interactions (3) via domain repeats (9) or specificity for more than one type of lipid (9,10).…”