Biochem. J. (1998) 332, 395–401 (Printed in Great Britain) 395 Characterization of the functional properties of smooth muscle caldesmon domain 4a : evidence for an independent inhibitory actin–tropomyosin binding domain Mohammed EL-MEZGUELDI 1 , O’Neal COPELAND, Iain D. C. FRASER, Steven B. MARSTON and Pia A. J. HUBER Cardiac Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, U.K. Recent analysis has shown the presence of three sequences in the C-terminal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin ATPase. Two are in domain 4b (amino acids 715–793) and one is in domain 4a (amino acids 636–714). In the present work we have compared recombinant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. We have produced three new recombinant fragments containing domain 4a : H10 [622–708], H12 [506–708] and H13 [622–726] and we have characterized their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the fragments H10, H12 and INTRODUCTION Caldesmon is a thin filament-associated protein first isolated from smooth muscle and later shown to be a component of a wide range of non-muscle cells [1]. It binds to many contractile proteins including actin, tropomyosin and myosin [2–4]. Caldesmon also binds to Ca+ -binding proteins such as calmodulin [2,5]. The binding of caldesmon to actin leads to the inhibition of the actin activation of myosin ATPase. This inhibition is greatly amplified by tropomyosin [6,7] and reversed by Ca+ -binding proteins at high Ca+ concentrations [8–10]. These properties have led to the proposal that caldesmon may regulate the thin filament interaction with myosin in smooth muscle and non-muscle cells [1,11]. An important feature in caldesmon regulatory function is that it is mediated by tropomyosin [12,13]. Caldesmon, at in io ratios to actin–tropomyosin, switches the thin filament between an ‘ on ’ and an ‘ off ’ state through a co-operative mechanism that is dependent on and propagated by tropomyosin [14]. Therefore definition of sites involved in actin binding and in the tropomyosin-dependent inhibition is essential for understanding caldesmon function. Caldesmon is a long molecule able to span 7 to 14 actin monomers when incorporated into the thin filament. In 1991, Marston and Redwood proposed a four-domain model for the caldesmon molecule [15]. There is general agreement that the inhibitory function of caldesmon is located near its C-terminus. All the actin-binding sequences and the entire tropomyosin- dependent inhibitory property are fully contained within the C- terminal 288 amino acids, approximately corresponding to domain 3 and 4 (reviewed in [16]). Many studies have shown that Abbreviations used : S-1, chymotryptic myosin subfragment ; DTT, dithiothreitol. 1 To whom correspondence and reprint requests should be addressed (e-mail : m.elmezgueldiic.ac.uk). H13 from actin. Thus the isolated caldesmon domain 4a peptides bind to the same region on actin as in the whole molecule while domains 4a and 4b occupy different sites on the actin molecule. Unlike domain 4b, none of the domain 4a fragments inhibited the actomyosin ATPase in the absence of tropomyosin. However both domain 4a and 4b fragments displayed an inhibitory activity in the presence of tropomyosin. H13 and H12 were more potent inhibitors than H10. Ca+ -calmodulin bound to H13 and reversed the inhibitory activity of this fragment but did not bind to H10 and H12. We conclude that domain 4a can act as an independent inhibitory actin–tropomyosin binding domain, but its properties are very different from the extreme C-terminal domain 4b. there is a minimal tropomyosin-dependent, Ca+ -calmodulin regulated actin inhibitory domain at the extreme C-terminus of caldesmon [17,18] ; the smallest C-terminal peptide reported to retain this function is 68 amino acids [19]. Within this short sequence, named domain 4b [18], two inhibitory actin binding sequences which overlap Ca+ -calmodulin binding sites have been identified [20,21]. Although isolated domain 4b contains a functional inhibitory property which resembles whole caldesmon it binds to actin relatively weakly and does not bind to tropomyosin at all [14,17–20,22]. In contrast, the binding of domain 4 as a whole quite closely resembles the intact caldesmon molecule [18,20], thus indicating that the N-terminal part of domain 4 (domain 4a) and perhaps parts of domain 3, contribute to caldesmon binding to actin and tropomyosin and to the consequent inhibition of the actomyosin ATPase. A number of authors have proposed actin binding sites within domain 4a and several peptides which contain domain 4a, but do not contain the extreme C-terminus have been shown to be tropomyosin dependent, Ca+ -calmodulin regulated inhibitors of actomyosin [20,23–25]. Moreover, Chalovich and co-workers [26] showed that a 7.3 kDa chymotryptic peptide corresponding to the human sequence 653–722 is able to inhibit actomyosin ATPase. However, large quantities of this peptide were needed to obtain maximum inhibition. In their recent paper, Fraser et al. [20] proposed that the actin binding site in domain 4a was one of three interacting segments needed for tropomyosin-dependent inhibition, but left open the question as to whether the segment in domain 4a had any autonomous inhibitory activity, as suggested by Chalovich et al. [26], or is only functional in concert with the segments in domain 4b.