Volume 20, Issue 11, Pages 1960-1970 (November 2012) Structures of the Phactr1 RPEL Domain and RPEL Motif Complexes with G-Actin Reveal the Molecular Basis for Actin Binding Cooperativity  Stephane Mouilleron, Maria Wiezlak, Nicola O’Reilly, Richard Treisman, Neil Q. McDonald  Structure  Volume 20, Issue 11, Pages 1960-1970 (November 2012) DOI: 10.1016/j.str.2012.08.031 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 An RPELPhactr1 Domain Helical Assembly Containing Three G-Actin Subunits (A) Domain structure of Phactr1 and MRTF-A. Basic regions (B1 and B2) contributing to a bipartite NLS are shown as black boxes and individual RPEL motifs as red boxes. (B) Structural alignment of RPEL domains from Phactr1 and MRTF-A. The basic regions are shown as gray shaded boxes, and RPEL motifs as pale red shaded boxes. Selected residues in green highlight key primary, secondary, and spacer interaction residues with G-actin. The location of each RPXXXEL motif is shown underneath the alignment. (C) Structure of the Phactr1 RPEL domain crank (red and white solid rendering) bound to three G-actin molecules (pale blue, green, and pink ribbon). The position of the screw axis is shown as a black line. (D) A difference mFo-DFc electron density map contoured at 3σ (left panel, shown in blue,) for the Phactr1 RPEL domain prior to its inclusion in refinement, overlaid on the refined crank-shaped RPEL domain structure. Right panel shows the structural superposition of the RPEL domain crank from Phactr1 (ribbon) and MRTF-A (solid) and their very similar trajectories. (E) Left panel, schematic of the trivalent Phactr1 and pentavalent MRTF-A RPEL domain assemblies with G-actin indicating the location of the first ”spacer” actin bound to MRTF-A. Right panel, the short spacer sequences within Phactr1 (gray) linking RPEL1 (pale blue) and RPEL2 (green) preclude binding of a spacer G-actin. Comparison with the MRTF-A spacer (yellow) connecting RPEL1 and RPEL2 (red) shows the G-actin cleft binding residues (yellow sticks) are missing in Phactr1. G-actin S1 is shown as a surface rendering with its hydrophobic cleft indicated (blue patch). See also Figures S1–S4 and Movies S1 and S2. Structure 2012 20, 1960-1970DOI: (10.1016/j.str.2012.08.031) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Primary G-Actin Contacts Made by RPELPhactr1 (A) Left panel: schematic showing the location of the primary G-actin binding sites within the trivalent RPELPhactr1 complex. The right panel shows a superposition of the three RPELPhactr1 motifs (colored according to left panel) onto G-actin R1 (pale gray, solid rendering). The panel reveals highly conserved primary actin interactions made by each RPEL. RPEL motif specific contacts are also shown. (B) Structural superposition of RPELPhactr1 peptides RPEL-N and RPEL3 (cartoon, deep pink, and orange) that bind to G-actin (pale gray, with the hydrophobic cleft and ledge interaction patches highlighted as per Figure 2A) with high affinity. Additional contacts unique to each of these RPEL motifs may contribute to their respective submicromolar affinities. See also Figures S5 and S6. Structure 2012 20, 1960-1970DOI: (10.1016/j.str.2012.08.031) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Secondary Actin Contacts within the G-Actin⋅RPELPhactr1 Helical Assembly (A) Left panel: schematic showing the location of the secondary G-actin binding sites within the trivalent RPELPhactr1 complex. Right panel: superposition of RPEL1:actin R2 and RPEL2:actin R3 showing selected secondary actin contacts as discussed in the text. RPEL1 (pale blue) and RPEL2 (green) are shown as cartoons and actins R1 and R2 are shown in pale gray surfaces (actin R1 hydrophobic cleft and ledge surfaces are colored as per Figure 2A). (B) Superposition of the secondary actin contacts made by the RPEL domain from Phactr1 (RPEL1 and RPEL2, colored as in [A]) and MRTF-A (RPEL1; pink) showing the strikingly similar contacts made by both proteins centered on the invariant RPEL motif glutamate. See also Figure S7. Structure 2012 20, 1960-1970DOI: (10.1016/j.str.2012.08.031) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 G-Actin⋅RPEL Peptidephactr1 Structures Form Open Helical Assemblies Containing the Secondary Actin Contacts (A) Filamental structures formed by single RPEL motifs (RPEL-N, left panel; RPEL2, right panel) within a crystal lattice. Consecutive adjacent asymmetric units each containing a single G-actin⋅RPEL motif peptide complex generate an open helical assembly, where each RPEL motif (cartoon) bridges two actin molecules (pale gray and white, respectively). The crystallographic 21 screw axis is shown as a black line. Actin cleft and ledge surfaces are colored as per Figure 2A. (B) Close-up of the secondary actin contacts made in RPEL-N and RPEL2 helical assemblies, centered on the conserved RPEL glutamate. RPEL motifs are drawn as cartoons and actin molecules as pale gray surfaces. Actin cleft and ledge surfaces are colored as per Figure 2A. (C) Cartoon depiction of the primary and secondary G-actin contacts made by an RPELPhactr1 motif, indicating the crucial contact residues observed. See also Figure S7. Structure 2012 20, 1960-1970DOI: (10.1016/j.str.2012.08.031) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Secondary Actin Contacts Are Required for Cooperative Actin-RPELPhactr1 Binding (A) Left panel: effects of RPEL mutations on primary actin binding affinities. The dissociation constants (Kd) were determined by fluorescence polarization anisotropy assay. Data for wild-type RPEL motifs and primary actin contact mutants R431ARPEL1, R469ARPEL2, and R507ARPEL3 are from Wiezlak et al (2012). ND, not detectable. Right panel: surface rendering of the RPEL-N peptide (orange surface) bound to the primary G-actin (white surface) and highlighting the secondary actin contacts (red surfaces). Actin hydrophobic cleft and ledge surfaces between actin subdomains 1 and 3 are colored as in Figure 2A. The secondary G-actin is omitted for clarity. Below, the RPEL-N primary sequence is shown with the primary and secondary contacts indicated. (B) Experimental molecular weights for RPELPhactr1 domain mutants derived from size exclusion chromatography coupled to multi-angle laser light scattering experiments (SEC-MALLS). Mutations are discussed in the text. Apparent stoichiometries from the molecular weights are shown in the right hand column. Structure 2012 20, 1960-1970DOI: (10.1016/j.str.2012.08.031) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 6 RPEL-N Secondary Contacts Are Required for Actin-Mediated Inhibition of Phactr1 Nuclear Import, Suggesting a Model for RPEL-N Recruitment into the RPEL Domain (A) RPEL-N putative secondary actin contacts are required for actin-mediated inhibition of serum-induced Phactr1 nuclear accumulation. The indicated FLAG-tagged Phactr1 derivatives were coexpressed with the nonpolymerizable actin R62D (Posern et al., 2002) in NIH 3T3 cells. We used a C-terminal truncation that ends at residue 528 (Phactr1 ΔC-terminus) for these experiments. Protein expression levels using this Phactr1 construct remain unaffected by R62D actin overexpression. The subcellular localization before and after 1 hr serum stimulation scored by immunofluorescence (C, cytoplasmic; N/C, pancellular; N, nuclear; at least 75 cells counted per point, error bars represent the SEM of three independent experiments). (B) A hypothetical model for the recruitment of G-actin⋅RPEL-N to the trivalent actin-bound RPELPhactr1 domain complex through RPEL-N secondary actin contacts to form a tetravalent nuclear import-inhibited G-actin⋅Phactr1 assembly. Structure 2012 20, 1960-1970DOI: (10.1016/j.str.2012.08.031) Copyright © 2012 Elsevier Ltd Terms and Conditions