The interface between MyBP-C and myosin: site-directed mutagenesis of the CX myosin-binding domain of MyBP-C C. A. MIYAMOTO 1,2 , D. A. FISCHMAN 2 and F. C. REINACH 1,2, * 1 Instituto de QuõÂmica, Universidade de Sa Äo Paulo, Caixa Postal 26077, CEP 05599-970, Sa Äo Paulo, S.P. Brazil; 2 Department of Cell Biology, Cornell University Medical College, 1300 York Ave, New York, NY 10021, USA Received 17 March 1999; accepted in revised form 4 August 1999 Abstract Myosin-binding protein-C (MyBP-C or C-protein) is a ca. 130 kDa protein present in the thick ®laments of all vertebrate striated muscle. The protein contains ten domains, each of ca. 90±100 amino acids; seven are members of the IgI family of proteins, three of the ®bronectin type III family. The motifs are arranged in the following order (from N- to C-terminus): Ig-Ig-Ig-Ig-Ig-Fn-Fn-Ig-Fn-Ig. The C-terminal Ig motif (domain X or CX) contains its light meromyosin-binding site. A recombinant form of CX, beginning at Met-1027, exhibits saturable binding to myosin with an anity comparable to the C-terminal 13 kDa chymotryptic fragment of native MyBP-C. To identify the surface in CX involved in its interaction with myosin, nine site-directed mutants (R37E, K43E, N49D, E52R, D56K, R73E, R74E, G80D and R103E) were constructed. Using a new assay for assessing the binding of CX with the light meromyosin (LMM) portion of myosin, we demonstrate that recombinant CX, just as the full-length protein, is able to facilitate LMM polymerization. Moreover, we show that residues Arg-37, Glu-52, Asp-56, Arg- 73, and Arg-74 are involved in this interaction with the myosin rod. All of these amino acids interact with negatively charged residues of LMM, since the mutants R37E, R73E and R74E are unable to bind myosin, whereas E52R and D56K bind myosin with higher anity than wild-type CX. Residues Lys-43 and Arg-103 show a small but signi®cant in¯uence on the binding reaction; residues Asn-49 and Gly-80 seem not to be involved in this interaction. Based on these data, a model is proposed for the interaction between MyBP-C CX and myosin ®laments. In this model, CX interacts with four molecules of LMM at four dierent sites of the binding protein, thus explaining the eects of MyBP-C on the critical concentration of myosin polymerization. Introduction MyBP-C (C-protein ca. 130 kDa), is a myosin-binding protein (Starr and Oer, 1971; Oer et al., 1973) localized to the central two-thirds of the cross-bridge bearing region (C-zone) of the A-bands of all vertebrates striated muscle (Bennett et al., 1985; Craig and Oer, 1976). It is distributed in 7±9 transverse stripes (the number varying with ®ber type) with a 43 nm axial spacing along the thick ®laments (Bennett et al., 1985; Craig and Oer, 1976; Dennis et al., 1984). The expres- sion of this protein is regulated during muscle develop- ment and is implicated in the assembly of thick ®laments in skeletal and cardiac muscles (Obinata et al., 1984; Lin et al., 1994). Analysis of the deduced primary structure suggests that MyBP-C is constituted of ten globular domains (numbered I to X), each ca. 90±100 amino acids in length, seven homologous to immunoglobulin (IgI) motifs and three to the ®bronectin (Fn) type III repeats (Einheber and Fischman, 1990; Okagaki et al., 1993). These domains are arranged in MyBP-C in the following sequence: Ig-Ig-Ig-Ig-Ig-Fn-Fn-Ig-Fn-Ig (Einheber and Fischman, 1990; Okagaki et al., 1993). The Ig motifs are homologous to telokin, the C-terminal domain of myosin light chain kinase (Holden et al., 1992). The functions of MyBP-C are uncertain. It interacts with the myosin rod in both its sub-fragment-2 (S2) and light meromyosin (LMM) domains (Moos et al., 1975; Starr and Oer, 1978; Gruen and Gautel, 1999). MyBP- C also binds titin (FuÈrst et al., 1992; Koretz et al., 1993; Freiburg and Gautel, 1996) and actin ®laments (Moos et al., 1978). Actin binding is inhibited by EGTA and restored with the addition of Ca 2+ (Moos, 1981; Yamamoto, 1986). MyBP-C is not required for either myosin or myosin rod polymerization in vitro. Puri®ed myosin forms ®laments with a 14.3 nm subunit period and a 43 nm axial repeat, although the ®laments usually vary in length and some exhibit a frayed appearance (Josephs and Harrington, 1966; Kaminer and Bell, 1966). When MyBP-C is added to the myosin solution before ®lament formation, depending on the molar ratio of MyBP-C to myosin, ®laments exhibit a dierent structure. At non- physiological molar ratios of MyBP-C:myosin ± either below or above physiological ratios ± there is no regularity of the myosin ®lament structure (Moos et al., *To whom correspondence should be addressed in Brazil: Fax: +55- 11-815-5579; E-mail: fdcreina@quim.ig.usp.br Journal of Muscle Research and Cell Motility 20: 703±715, 1999. 703 Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.