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

Cyclase-associated protein (CAP) is a conserved actin-binding that regulates multiple aspects of actin dynamics, including polymerization, depolymerization, filament severing, and nucleotide exchange. CAP has been isolated from different cells tissues in an equimolar complex with actin, previous studies have shown CAP–actin contains six molecules each actin. Intriguingly, here, we successfully Xenopus cyclase-associated 1 (XCAP1) oocyte extracts, which contained only four XCAP1 This XCAP1–actin remained stable as single population 340 kDa species during hydrodynamic analyses using gel filtration or analytical ultracentrifugation. Examination the by high-speed atomic force microscopy revealed tripartite structure: one middle globular domain two arms. The arms were observed high low states. at state spontaneously converted to dissociation complex. However, when extra G-actin was added, state. Based on known structures N-terminal helical-folded C-terminal CARP domain, hypothesize corresponds tetramer arm heterotetramer containing dimer molecules. novel configuration should help understand how promotes disassembly. Regulated assembly disassembly filaments are vital diverse function cytoskeleton (1Pollard T.D. Cooper J.A. Actin, central player cell shape movement.Science. 2009; 326: 1208-1212Crossref PubMed Scopus (1265) Google Scholar). actin-regulatory proteins control key dynamics (2Ono S. role regulating - more than monomer-sequestration factor.J. Cell Sci. 2013; 126: 3249-3258Crossref (73) Scholar, 3Rust M.B. Khudayberdiev Pelucchi Marcello E. CAPt’n dynamics: Recent advances molecular, developmental physiological functions (CAP).Front. Dev. Biol. 2020; 8: 586631Crossref (7) originally identified yeast binds adenylyl cyclase involved Ras signaling pathway (4Field J. Vojtek A. Ballester R. Bolger G. Colicelli Ferguson K. Gerst Kataoka T. Michaeli Powers Riggs M. 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When applied column glutathione S-transferase (GST)-fused ADF/cofilin (XAC) immobilized, several specifically bound described previously (35Okada Obinata Abe XAIP1: homologue interacting (AIP1), induces cooperatively family proteins.J. 1999; 112: 1553-1565Crossref 1A). 65-kDa, 42-kDa, 19-kDa actin-interacting (XAIP1), XAC, respectively Peptide sequencing 94-kDa 60-kDa gelsolin (36Ankenbauer Kleinschmidt Vandekerckhove Franke W.W. Proteins oogenesis early embryogenesis laevis: Gelsolin major cytoplasmic 1988; 107: 1489-1498Crossref (37KhosrowShahian Hubberstey A.V. Crawford M.J. expression developmentally regulated Xenopus.Mech. 113: 211-214Crossref (4) respectively. attempted isolate anion-exchange chromatography followed hydroxyapatite chromatography, not separated these procedures together 1B). Further Sephadex G-200 resulted coelution peak ?390 (our unpublished observation), much larger alone, 1:1 indicating they Native molecular mass determined accurately methods: size-exclusion coupled multiangle light scattering (SEC-MALS) SEC-MALS, resolved 1C). There no detectable peaks corresponded SEC-MALS analysis. Likewise, ultracentrifugation, 337 (S = 10) 1D), agrees result SEC-MALS. Considering masses individual (52 kDa) (42 kDa), closely matched (calculated 376 kDa). experimentally ?10% smaller calculated mass. could due partial analyses, limitation some cases experiments (38Folta-Stogniew Williams K.R. Determination solution: Implementation HPLC exclusion laser service core laboratory.J. Biomol. Tech. 51-63PubMed Since bind monomers, likely G-actin. results indicate ratio. examined (HS-AFM) (Figs. 3). Typical images mica surfaces showed consisted three domains 2A), designated (MGD, red Fig. 2A cartoons) (Arm Arm green blue cartoons). height MGD 3.6 ± 0.9 nm (n 107) B C) relatively time-lapse imaging G, Supplementary Movie S1). By contrast, states: (Arm-HS, (Arm-LS, cartoons, see Arm-HS 7.5 0.5 855) D E), while Arm-LS 3.3 0.3 1078) 2F 3, C D). cases, transitioned either line ?0.9 s) 4.5 s), suggesting association component, presumably G-actin, occurred observations. Over periods HS-AFM observations, gradually decreased, predominated, over time repeated tapping AFM probe adsorption surface (see below). Some had throughout observations 2A, indicated dashed line, 3A, S2), B–D).Figure 3The both stable. A, S2). Scanning area 80 × 64 nm2 48 pixels. Imaging rate 66 ms/frame (?15 fps). Bar, 20 nm. B, course heights domains. C–F, cross-sectional (C, green) (E, red) straight colored lines drawn image Height distributions (D) (F) Gaussian fitting yielded average figure. G–I, distances their highest points (G). Distribution distance (H) (I) figure.View Large Image Figure ViewerDownload Hi-res Download (PPT) very dynamic S1), restricted within 14.8 4.9 214) H I) if connected flexible linkers. fluctuated wide 17.4 7.6 427) J), further supporting linkers arm. once Arm-LS, stabilized A–D, unchanged E F). became wider [22.9 5.7 539)], whereas narrower [13.0 3.7 1078)]. These suggest physically surface. To test features used treated APTES, adds positive charges nonspecific strong proteins. On APTES-treated mica, almost always detected (Arm-LS) 3.0 0.4 2293) 4, S3). indistinguishable normal E, constant 25.4 5.6 1331) G H), 2I), strongly immobilized spread apart. manner 3I). onto solid artificially converts causing arm-bound conversion association, effects additional free ADP-G-actin 5). Freshly prepared samples mostly 5A). after 15 min, them 5B). After (after ?20 min), final 100 nM 5, ADP-G-actin, frequent reversible conversions 5C, S4). example 5C (dashed circle), initially (green arrowheads) 0.6 s top left panel), then (blue arrowhead) 0.8 second another 2.2 fourth panel). Conversely, right panel 10.6 bottom 11.6 s). plot 5D) indicates independently coordination 0.14 0.04 molecules?1 s?1 5E). Even absence occasionally 0.03 5E) rebinding consistent spontaneous transition 1A. alone objects (N.D.: none detected, component biochemical biophysical properties other species, propose model appearance “butterflies” (CARP/G-actin) “flower” 6). HFD (Arm-HS) itself Dictyostel