Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Abstract

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca(2+)-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with Kd values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.

Bridging of membrane surfaces by annexin A2

“…This complex contains two membrane binding AnxA2 moieties and has been shown to bridge membrane surfaces by cryo-electron microscopy 25 . Addition of 50 nM A2t in the presence of 250 µM Ca 2+ yielded frequency and dissipation shifts of ΔF 3 = −67.5+ /− 0.05 Hz and ΔD 3 = 1.38+ /− 0.02 * 10 −6 (step 2) that are in line with previously reported values 26 and indicative of high affinity protein adsorption to the SLB. After a steady state equilibrium was reached, LUVs were added in Ca 2+ -containing HBS buffer (step 3) leading to an additional frequency shift of ΔF 3 = −279.1+ /− 0.1 Hz with a distinct dissipation shift of ΔD 3 = 24.8+ /− 0.06 * 10 −6 .…”

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

“…The resulting dampening of the quartz sensor oscillation is referred to as dissipation (D). The monitored dissipation changes (ΔD) correlate with the viscoelastic properties of the bound mass 18 and are defined as follows 8 .…”

Receptor‐Free Signaling at Curved Cellular Membranes

“…[5,62,63] Within the framework of curvature-sensing proteins, lipid dependence has been demonstrated for the BAR domain proteins Pacsin 1, Pacsin 2, and FBP17 (to PS or PIP and PS [59] ). [5,62,63] Within the framework of curvature-sensing proteins, lipid dependence has been demonstrated for the BAR domain proteins Pacsin 1, Pacsin 2, and FBP17 (to PS or PIP and PS [59] ).…”