Structural insights into the inhibition of actin-capping protein by interactions with phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate

Abstract

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP(2)). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP(2)/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP-membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.

Figure 1. Phylogenetic analysis of CPα (A) and CPβ (B).

Both trees represent protein bayesian phylogeny of particular genes. Numbers at nodes correspond to posterior probabilities from Bayesian analysis and the approximate likelihood ratio test with SH-like (Shimodaira-Hasegawa-like) support from maximum likelihood method, respectively. Circles represent support 100% by both methods, Missing values indicate support below 50%, dash indicates that a different topology was inferred by ML method. Branches were collapsed if inferred topology was not supported by both methods. Scale bar indicating the rates of substitutions/site is shown in corresponding tree. Abbreviations used: Agos – Ashbya gossypii, Alyr – Arabidopsis lyrata, Anig – Aspergillus niger Atha – Arabidopsis thaliana, Bden – Batrachochytrium dendrobatidis, Calb – Candida albicans, Ccin – Coprinopsis cinerea, Cele – Caenorhabditis elegans, Cint – Ciona intestinalis, Cneo – Cryptococcus neoformans, Cpos – Coccidioides posadasii, Ddis – Dictyostelium discoideum, Dhan – Debaryomyces hansenii, Dmel – Drosophila melanogaster, Dpur – Dictyostelium purpureum, Dpul – Daphnia pulex, Drer - Danio rerio, Ehis - Entamoeba histolytica, Ehux - Emiliania huxleyi, Ggal – Gallus gallus, Gzea – Gibberella zeae, Hsap – Homo sapiens, Mbre – Monosiga brevicollis, Mcir – Mucor circinelloides, Mmus – Mus musculus, Ngru – Naegleria gruberi, Ntab – Nicotiana tabacum, Nvec – Nematostella vectensis, Osat – Oryza sativa, Pbla – Phycomyces blakesleeanus, Pfal – Plasmodium falciparum, Phtr – Phaeodactylum tricornutum, Ppat – Physcomitrella patens, Pram – Phytophthora ramorum, Psoj – Phytophthora sojae, Ptet - Paramecium tetraurelia, Ptri – Populus trichocarpa, Pviv – Plasmodium vivax, Sbic – Sorghum bicolor, Scer – Saccharomyces cerevisiae, Slyc – Solanum lycopersicum, Spom – Schizosaccharomyces pombe, Smoe – Selaginella moellendorffi, Tadh – Trichoplax adherans, Tcru – Trypanosoma cruzi, Trub – Takifugu rubripes, Tthe – Tetrahymena thermophila, Tvag – Trichomonas vaginalis, Umay – Ustilago_maydis and Vvin – Vitis vinifera.

Figure 2. Structural comparison of AtCP and GgCP.

A Superimposition of the homology-model for plant AtCP (in green) on the X-ray structure of chicken GgCP (in blue). B Electrostatic potential mapped on the structure of AtCP and GgCP ranging from −5 (red) to +5 (blue) kbT/e_c. This figure was prepared with the UCSF Chimera package .

Figure 3. Self-assembly of lipid bilayer in the presence of AtCP.

Self-assembly CG-MD simulation of membrane containing 20% POPA (charge −2)/POPC at time A 0 ns, B 5 ns, C 20 ns, and D 100 ns. CG water molecules and Na⁺ ions are not shown for the sake of clarity. Headgroups and glycerol backbone atoms of POPA are highlighted in van der Waals representation. Only protein backbone atoms are shown in licorice representation. This figure was prepared using VMD .

Figure 4. Comparison of interaction of AtCP and GgCP with distinct membranes at 500 ns.

Chemical diagrams and CG representations of A POPA and D POPIP₂. The final state of the MD system containing B AtCP – 20% POPA (charge −2)/POPC, C GgCP – 20% POPA (charge −2)/POPC, E AtCP – 5% POPIP₂/POPC and F GgCP – 5% POPIP₂/POPC. CG water molecules and Na⁺ ions are not shown for the sake of clarity. Headgroups and glycerol backbone atoms of POPIP₂ and POPA are highlighted in van der Waals representation. AtCP is colored green and GgCP is blue; only backbone atoms are shown in licorice representation. This figure was prepared with VMD .

Figure 5. Polar and nonpolar contacts of AtCP (A,B) and GgCP (C,D) with distinct membranes.

Polar contacts were defined as the number of POPA/POPIP₂ headgroup atoms within 8 Å of protein atoms. Nonpolar contacts were defined as the number of POPA/POPIP₂ and POPC tail atoms within 8 Å of protein atoms. Contacts represent the average number computed for each performed simulation over last 200 ns. This figure was prepared using VMD .

Figure 6. Effects of mutations in AtCP on its membrane association.

Time-course for three independent simulations with wild-type (WT) AtCP and several different mutations is shown as the distance of the center of mass of the protein from the center of mass of the bilayer. A System with 20% POPA/POPC. B System with 5% POPIP₂/POPC.

Figure 7. Potential of mean force (PMF) curves for pulling AtCP (A) and GgCP (B) from distinct membranes.

Red lines represent PMF curves for pulling respective protein from membranes containing 20% POPA/POPC. Blue lines represent PMF curves for pulling respective protein from membranes containing 5% POPIP₂/POPC. Vertical red and blue lines indicate error bars generated by the Bayesian boostrap method of g_wham program .

Figure 8. Details in the interaction of AtCP with the membrane containing phosphatidic acid.

A Sequence comparison of C-terminal parts of CPα (CPα-Cterm) from different species. The mafft algorith was used to construct multiple alignments and the final figure was produced using the Jalview alignment editor . Abbreviations used: At – Arabidopsis thaliana, Gg – Gallus gallus, Hs – Homo sapiens, Mb - Monosiga brevicollis, Os – Oryza sativa, Pp – Physcomitrella patens, Sc - Saccharomyces cerevisiae, Sm - Selaginella moellendorffii, Sp - Schizosaccharomyces pombe. Red asterisk marks conserved Lys in plants. B A detailed view of AtCP interaction with membrane containing 20% POPA (charge −2)/POPC. This figure was prepared using VMD . C Protein-lipid overlay assay for detecting interacting lipids. CPα-Cterm shows a preference for PA and PPIs. GST-CPα-Cterm bound to the lipids was detected by immunoblotting with an antibody against GST. Figure shows a representative result from 3 different experiments. D Liposome-binding assay of CPα-Cterm. PA binding was determined using 200 nm-sized vesicles containing 20% PA/PC or PC alone. After incubation of GST-AtCPα-Cterm with the vesicles, they were recovered by ultracentrifugation and protein bound was analysed by SDS-PAGE. As negative control, GST alone was used. Figure shows representative result from 4 different experiments.