Structure and function of palladin's actin binding domain

Abstract

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.

Keywords: 3D; 4′,6-diamidino-2-phenylindole; ABP; DAPI; F-actin; FLNa; G-actin; GFP; HSQC; PDB; Protein Data Bank; RASREC; VASP; actin binding protein; actin-binding protein; crosslinking; electrostatics; filamentous actin; filamin A; globular or monomeric actin; green fluorescent protein; heteronuclear single quantum correlation; immunoglubulin-like domain; palladin; resolution-adapted structural recombination; three-dimensional; vasodilator-stimulated phosphoprotein.

Figure 1

NMR structure of actin-binding domain of palladin (a) Ribbon diagram of M. musculus palladin Ig3 domain, lowest energy structure from CYANA-CS-Rosetta (PDB entry 2LQR). Nine β-strands are colored from N to C termini (A, red; A’, orange; B, yellow; C, green; C’, dark green; D, cyan; E, blue; F, magenta; G, purple) (b) Overlay of 20 lowest energy structures, with calculated RMSD for all ordered residues (6–100), backbone only = 0.7 and all heavy= 1.0. (c) Surface electrostatic potential of two faces of Ig3, highlighting two basic patches containing either K51 or K15/K18 (blue, positive charge; red, negative charge; and white, neutral). Charged surfaces calculated using PDB2PQR Server.

Figure 2

Basic patches on Ig3 surface are critical for binding F-actin. (a) Results of binding co-sedimentation assays with F-actin, including control experiments indicated by * (actin, K15/18A, and WT Ig3 alone). Supernatant (S) and pellet (P) fractions were analyzed by gel electrophoresis. Wildtype and lysine point mutants of the Ig3 domain were purified and assayed for actin binding by co-sedimentation. Mutations of the lysine residues at residues 15, 18, and 51 all significantly reduced binding to F-actin. (b) Quantitative analysis of the amount of Ig3 bound in the presence of WT and mutant Ig3 domains. For all co-sedimentation assays the Ig3 concentration was 10 µM and actin concentration was 20 µM, and each experiment was repeated at least three times with standard deviation error bars shown.

Figure 3

F-actin bundling by Ig3 domain of palladin. Increasing the concentration of Ig3 relative to F-actin revealed that this domain is capable of bundling actin. (a) Differential centrifugation assay was used to assess crosslinking by WT Ig3, where the samples were subjected to a low speed spin, and actin bundles (B) in the pellet were collected before sedimenting the remaining F-actin by ultracentrifugation and resolving the supernatant (S) and pellet (P) from these spins by SDS-PAGE. (b) Quantification of actin bundling was analyzed by densitometry of the actin bands to estimate the ability of palladin to bundle F-actin. In all assays, the concentration of F-actin was held constant at 10 µM and the palladin was varied from 10 µM to 40 µM. The percent of actin found in the low speed bundle is represented by the black bar, soluble portion is white, and high speed pelleted actin is gray. (c) Wildtype and mutant forms of Ig3 domain were also assessed for actin crosslinking using the same differential centrifugation assay. Bundling assays carried out with basic lysine residues mutations (K15/18A or K51A) of Ig3 eliminate F-actin bundling by palladin. (d) Quantitative analysis of the amount of actin found in the bundle, supernatant and pellet in the presence of WT and mutant Ig3 domains. Data are means ± standard deviation for three or more separate measurements.

Figure 4

Ig3 domain of palladin plays a critical role in subcellular targeting. Full-length palladin (WT), single, double and triple point mutants (K51A, K15/18A, and K15/18/51A) and Ig3 deletion mutant (ΔIg3) were introduced into a plasmid encoding full-length GFP-tagged palladin and transfected into cultured COS-7 cells. 24 hours after transfection F-actin was stained with Alexafluor 568 phalloidin and GFP-tagged palladin variants were analyzed by fluorescence microscopy. DAPI staining was used to detect the nuclei.