Coarse-grained modeling of annexin A2-induced microdomain formation on a vesicle

Abstract

Annexin A2 (A2)-induced microdomain formation is a key step in biological processes such as Ca²⁺-mediated exocytosis in neuroendocrine cells. In this work, a total of 15 coarse-grained molecular dynamics simulations were performed on vesicle models having a diameter of approximately 250 Å for 15 μs each using the Martini2 force field. Five simulations were performed in the presence of 10 A2, 5 in the presence of A2 but absence of PIP₂, and 5 simulations in the absence of A2 but presence of PIP₂. Consistent results were generated among the simulations. A2-induced PIP₂ microdomain formation was observed and shown to occur in three phases: A2-vesicle association, localized A2-induced PIP₂ clustering, and A2 aggregation driving PIP₂ microdomain formation. The relationship between A2 aggregation and PIP₂ microdomain formation was quantitatively described using a novel method which calculated the variance among protein and lipid positions via the Fréchet mean. A large reduction in PIP₂ variance was observed in the presence of A2 but not in its absence. This reduction in PIP₂ variance was proportional to the reduction observed in A2 variance and demonstrates that the observed PIP₂ microdomain formation is dependent upon A2 aggregation. The three-phase model of A2-induced microdomain formation generated in this work will serve as a valuable guide for further experimental studies and the development of novel A2 inhibitors. No microdomain formation was observed in the absence of A2 and minimal A2-membrane interaction was observed in the absence of PIP₂.

Figure 1

Colored and labeled cartoon representations of Annexin A2 structural features. (A) An Annexin A2 repeat and with the A, B, C, D, and E labeled. (B) The full Annexin A2 structure where the ND, RI, RII, RIII, and RIV, are colored and labeled. To see this figure in color, go online.

Figure 2

Graphical representations of the simulation setups. Presolvation structures of the (A) PIP₂⁺-A2V, (B) PIP₂^–-A2V, and (C) PIP₂⁺-V systems. (D) Cross section of the solvated PIP₂⁺-A2V system. All components of the system are represented using spheres. The color key for all systems follows: A2, gray; POPC, blue; POPS, light green; PIP₂, red; CHL, tan; water, teal; Na⁺, magenta; Cl⁻, light orange. To see this figure in color, go online.

Figure 3

Key A2 and PIP₂ clustering events. (A1–A4) Snapshots of A2-I from simulation run 1 of the A2V system highlighting the progressive formation of a PIP₂ cluster underneath A2-I over the course of ∼1 μs of the simulation. Labels identify the time and number of PIP₂ contacts with A2-I. (B1–B5) Snapshots of the largest A2 aggregate in the final frame of A2V simulation runs 1–5, respectively. (C1–C2) The final frame from simulation run 1 of the A2V simulation (C1) without A2 shown and (C2) with A2 shown. (C3) Final frame from the vesicle simulation. In all images POPC, POPS, PIP₂, and CHL are colored purple, green, red, and tan, respectively. In (A1–A4) A2 are rendered using a transparent material to enhance visibility of the PIP₂ cluster beneath. In (B1–B4, C1–C3) A2 are colored gold, green, or light blue such that they are easily distinguishable from their neighbors. To see this figure in color, go online.

Figure 4

3D representations of contact analysis performed for the PIP₂⁺-A2V system. (A) Views of A2 models with each residue colored according to its percent contribution to total A2-A2 contacts. Labels are shown for residues that contribute more than 1.5% of all A2-A2 contacts. (B) View of an A2 model’s convex face with residues colored according to their percent contribution to total A2-membrane contacts. Labels are shown for residues that contribute more than 2.0% of all A2-membrane contacts. (C) Views of an A2 model are show with residues experimentally identified as important for A2 aggregation via cross-linking studies represented as red spheres and the top 10 residues we identified as important for A2-A2 contacts colored blue. The provided color bar corresponds to both (A) and (B). Repeats of A2 are labeled in all images. To see this figure in color, go online.

Figure 5

Lipid and A2 variance plots. (A) Plot of A2 and PIP₂ variances versus time with separate y axes scales specified. (B) Plot of PIP₂ variance versus A2 variance with a trendline included. Variance for all lipids from (C) the V system, which lacks A2, and (D) the A2V system, which contains A2. To see this figure in color, go online.

Figure 6

Graphical summary of A2-induced PIP₂ microdomain formation. POPC, POPS, and PIP₂ lipid heads are colored purple, green, and red, respectively. Lipid tails are colored gray. A2 is colored tan and represented as a cartoon. The first image represents the initial association of A2 with the membrane, the second image represents A2-induced local aggregation of PIP₂, the final image represents A2 aggregation and the associated formation of PIP₂ microdomains. To see this figure in color, go online.