Native cyclase-associated protein and actin from Xenopus laevis oocytes form a unique 4:4 complex with a tripartite structure

Abstract

Cyclase-associated protein (CAP) is a conserved actin-binding protein that regulates multiple aspects of actin dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. CAP has been isolated from different cells and tissues in an equimolar complex with actin, and previous studies have shown that a CAP-actin complex contains six molecules each of CAP and actin. Intriguingly, here, we successfully isolated a complex of Xenopus cyclase-associated protein 1 (XCAP1) with actin from oocyte extracts, which contained only four molecules each of XCAP1 and actin. This XCAP1-actin complex remained stable as a single population of 340 kDa species during hydrodynamic analyses using gel filtration or analytical ultracentrifugation. Examination of the XCAP1-actin complex by high-speed atomic force microscopy revealed a tripartite structure: one middle globular domain and two globular arms. The two arms were observed in high and low states. The arms at the high state were spontaneously converted to the low state by dissociation of actin from the complex. However, when extra G-actin was added, the arms at the low state were converted to the high state. Based on the known structures of the N-terminal helical-folded domain and C-terminal CARP domain, we hypothesize that the middle globular domain corresponds to a tetramer of the N-terminal helical-folded domain of XCAP1 and that each arm in the high state corresponds to a heterotetramer containing a dimer of the C-terminal CARP domain of XCAP1 and two G-actin molecules. This novel configuration of a CAP-actin complex should help to understand how CAP promotes actin filament disassembly.

Keywords: Xenopus; actin; atomic force microscopy (AFM); cyclase-associated protein; cytoskeleton; protein complex.

Figure 1

Determination of native molecular weight of the XCAP1–actin complex.A and B, purification of XCAP1–actin complex from Xenopus oocyte extracts. A, proteins that bound to the XAC-affinity column were eluted, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Each band was identified by peptide sequencing as shown on the right of the gel. B, a complex of XCAP1 and actin was isolated after anion-exchange chromatography and hydroxyapatite chromatography. Positions of molecular mass markers in kDa are shown on the left. C, SEC-MALS analysis of the XCAP1–actin complex. Purified XCAP1–actin complex was applied to size-exclusion chromatography, and refractive index (mV, red), right-angle light scattering (mV, dark green), low-angle light scattering (mV, black), MALS signal at 90° (mV, light green) were monitored. D, analytical ultracentrifugation analysis of the XCAP1–actin complex. A single peak of 10S was detected indicating that the XCAP1–actin complex was stable.

Figure 2

High-speed atomic force microscopy reveals a tripartite structure of the XCAP1–actin complex.A, time-lapse HS-AFM images of the XCAP1–actin complex on a mica surface (see Supplementary Movie S1). Scanning area was 80 × 64 nm² with 64 × 48 pixels. Imaging rate was 66 ms/frame (∼15 fps). Bar, 20 nm. Schematic representation of molecular features is shown in the bottom panels: middle globular domain (MGD, red), arm in the low state (Arm-LS, green), and arm in the high state (Arm-HS, blue). The complex indicated by dashed lines in the second frame had both arms in Arm-LS throughout the observation (see Fig. 3 for quantitative analysis of Arm-LS). B–F, cross-sectional analyses of MGD (B, red), Arm-HS (D, blue), and Arm-LS (F, green) at the straight colored lines drawn on the images in A. Height distributions of MGD (C) and Arm-HS (E) and single Gaussian fitting yielded average heights of MGD and Arm-HS as indicated in the figure. G, time course of the heights of three globular domains. Green-shaded areas indicate periods when one of the arms was in the low state. H–J, time course of the distances between the domains at their highest points (H). Distribution of the distance between MGD and one of the arms (I) and between two arms (J) and single Gaussian fitting yielded average distances as indicated in the figure. Arm-HS was selected in these analyses.

Figure 3

The XCAP1–actin complex with both arms in a low state is stable.A, time-lapse HS-AFM images of the XCAP1–actin complex containing both arms in Arm-LS on a mica surface (see Supplementary Movie S2). Scanning area was 80 × 64 nm² with 64 × 48 pixels. Imaging rate was 66 ms/frame (∼15 fps). Bar, 20 nm. B, time course of the heights of three globular domains. C–F, cross-sectional analyses of Arm-LS (C, green) and MGD (E, red) at the straight colored lines drawn on the image in A. Height distributions of Arm-LS (D) and MGD (F) and single Gaussian fitting yielded average heights of Arm-LS and MGD as indicated in the figure. G–I, time course of the distances between the domains at their highest points (G). Distribution of the distance between two arms (H) and between MGD and one of the arms (I) and single Gaussian fitting yielded average distances as indicated in the figure.

Figure 4

Strong adsorption of the XCAP1–actin complex to a charged surface stabilizes two arms in a low state.A, time-lapse HS-AFM images of the XCAP1–actin complex on an APTES-treated mica surface (see Supplementary Movie S3). Scanning area was 100 × 100 nm² with 80 × 80 pixels. Imaging rate was 100 ms/frame (10 fps). Bar, 20 nm. B, time course of the heights of three globular domains. C–F, cross-sectional analyses of Arm-LS (C, red) and Arm-LS (E, green) at the straight colored lines drawn on the image in A. Height distributions of Arm-LS (D) and MGD (F) and single Gaussian fitting yielded average heights of MGD and Arm-HS as indicated in the figure. G–I, time course of the distances between the domains at their highest points (G). Distribution of the distance between two arms (H) and between MGD and one of the arms (I) and single Gaussian fitting yielded average distances as indicated in the figure.

Figure 5

Addition of ADP-G-actin to the XCAP1–actin complex promotes conversion of arms from a low to high state.A and B, representative images of the XCAP1–actin complex on a mica surface immediately after observation (A) and after 15 min of observation (B). C–E, time-lapse HS-AFM images of the XCAP1–actin complex after addition of final 100 nM ADP-G-actin (see Supplementary Movie S4). The complex indicated by a dashed circle showed frequent and reversible conversions between Arm-LS (green arrowheads) and Arm-HS (blue arrowheads) as plotted in (D). Red arrowheads in (D) indicate conversion events from Arm-LS to Arm-HS. E, quantitative analysis of frequency of binding events (conversion from Arm-LS to Arm-HS; molecules⁻¹ s⁻¹). In the presence of ADP-G-actin only and in the absence of the XCAP1–actin complex and ADP-G-actin, no objects that matched the size of Arm-LS or Arm-HS were observed, (N. D., none detected). Sample numbers are shown in the brackets.

Figure 6

Model of the CAP–actin complex.A, domain structure of CAP [adopted from (2)]. Approximate molecular masses of segments are shown. B, a model of the CAP–actin complex. Crystal structures of HFD of mouse CAP1 (Protein Data Bank accession ID: 6RSQ) and CARP domain of mouse CAP1 bound to actin (Protein Data Bank accession ID: 6FM2) were used to reconstruct a CAP–actin complex at a 4:4 stoichiometric ratio. Putative locations of oligomerization motif and flexible linkers are indicated by dashed lines.