Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin–actin interaction Imen Ferjani a,d , Abdellatif Fattoum c , Mohamed Manai d , Yves Benyamin a , Claude Roustan a , Sutherland K. Maciver b, ⁎ a UMR 5539 (CNRS) Laboratoire de motilité cellulaire (Ecole Pratique des Hautes Etudes) Université de Montpellier 2, Place E. Bataillon, CC107, 34095 Montpellier Cedex 5, France b Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD Scotland, UK c Centre de Recherches de Biochimie Macromoléculaire, FRE 2593 (CNRS), 1919 rte de Mende, 34293 Montpellier Cedex 5, France d Unité de Biochimie et Biologie Moléculaire, Faculté des Sciences de Tunis, Campus Universitaire 2092 El Manar, Tunis, Tunisie abstract article info Article history: Received 3 December 2009 Received in revised form 5 May 2010 Accepted 25 May 2010 Available online 1 June 2010 Keywords: Actin cytoskeleton Calponin Microfilaments CH domain Spectrin α-Actinin Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478–485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18–28 and 360–372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the α-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. 1. Introduction Actin is a major component of the cytoskeleton and is the principle factor in determining cell structure, shape, and motility of the typical eukaryotic cell. The actin filament network is modulated by a host of actin-binding proteins [1] such as the calponins, a family that are expressed widely in vertebrate cells [2–4]. Calponins are thought to be involved in important physiological activities including regulation of acto-myosin ATPase activity [5], structural organization of cytoskel- eton [6,7], and cell signaling at cell membranes [8,9]. Calponin is known to bind phospholipids in vitro [10] and appears to be associated with the plasma membrane in a variety of cell types [3,11], possibly as a complex with the actin filament severing protein gelsolin [12,13]. Calponin has given its name to a motif known as the “calponin homology” domain (CH domain) found in a wide variety of actin- binding proteins [14]. This is despite the fact that the CH domain of calponin itself does not bind actin [15]. A single CH domain is found in Vav [16] and other proteins [17]. Tandem CH domains (CH1 and CH2) are found in the spectrin superfamily of actin-binding proteins that includes utrophin, dystrophin, α-actinin, ABP120, and filamin [17,18], and the fimbrin/plastin family of actin cross-linking molecules contains four copies of the CH domains [19,20]. Three distinct actin- binding sites (ABS1–3) have been identified within the tandem CH domains [17]. ABS1 was identified in α-actinin [21] and in dystrophin [22]. A 27-amino acid region that straddles the final helix of CH1 and the linking region to CH2 of ABP120 from Dictyostelium has been identified as an actin-binding region [23]. A homologous region that binds actin has been identified in α-actinin [24] and dystrophin [22] and this region (now known as ABS2) is conserved amongst other spectrin superfamily members CH tandems but not to the CH domains of Vav or calponin [25]. A third site ABS3 has also been identified [22]. ABS2 is homologous to part of the actin-binding region further toward the C-terminus of calponin (Table 1). Calponin has also given its name Biochimica et Biophysica Acta 1804 (2010) 1760–1767 Abbreviations: CH, calponin homology domain; CLIK, calponin-like repeat; acrylodan, 6-acryloyl-2-dimethylaminonaphthalene; AEDANS, N,-iodoacetyl-N′-[sul- pho-1-naphthyl]-ethylenediamine; FITC, fluorescein 5-isothiocyanate ⁎ Corresponding author. E-mail address: smaciver@staffmail.ed.ac.uk (S.K. Maciver). 1570-9639/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.05.012 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap