Crystallographic analysis of calcium-dependent heparin binding to annexin A2

Abstract

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

FIGURE 1. Structural alignment (backbone representation) of annexin A2 structures; uncomplexed annexin A2 (red), tetrasaccharide-bound annexin A2 (green), and hexasaccharide-bound annexin A2 (cyan)

Structures are shown in wall-eyed stereo view. Blue spheres depict Ca²⁺ ions unique to the uncomplexed annexin A2 structure; yellow spheres indicate Ca-8; blue/yellow spheres indicate Ca²⁺ ions present in all structures. For clarity, only the oligosaccharides from the hexasaccharide complex (cyan, labeled as HHS) are shown.

FIGURE 2. Structures and electron density of the heparin tetrasaccharide (A) and hexasaccharide (B) bound to annexin A2

Electron density, contoured at 1σ, is from simulated-annealing omit map omitting the oligosaccharides, Ca-7 and Ca-8. Structures are shown in wall-eyed stereo view. Domains I and IV are colored orange and magenta, respectively. Yellow spheres denote Ca²⁺. Red dashed lines represent polar contacts between annexin A2 and oligosaccharides or Ca-8, as listed in Table 2. Heparin oligosaccharide residues from left (nonreducing end) to right are designated as: A, Δ⁴UAp2S; B, GlcNpS6S; C, IdoAp2S; D, GlcNpS6S; E, IdoAp2S.

FIGURE 3. Calcium coordination at the heparin-binding site

Structure is displayed in wall-eyed stereo view. Ca²⁺ ions are shown as yellow spheres and water molecules as red spheres. Orange dashed lines denote Ca²⁺ coordination bonds and interactions between water molecules and oligosaccharide. All distances between Ca²⁺ ions and oxygen ligands fall between 2.3 and 2.6 Å.

FIGURE 4. SPR analysis of the annexin A2 interaction with heparin polysaccharide

a, sensorgrams of annexin A2-heparin interaction at pH 7.4. Annexin A2 concentrations (from top to bottom): 500, 200, 100, 50, and 10 n^M. b, sensorgrams of annexin A2-heparin interaction at pH 5.0. Annexin A2 concentrations (from top to bottom): 4000, 2000, 1000, and 500 n^M.