The interface between MyBP-C and myosin: site-directed mutagenesis of the CX myosin-binding domain of MyBP-C.
Myosin-binding protein-C (MyBP-C or C-protein) is a ca. 130 kDa protein present in the thick filaments 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 fibronectin 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 affinity 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 affinity than wild-type CX. Residues Lys-43 and Arg-103 show a small but significant influence 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 filaments. In this model, CX interacts with four molecules of LMM at four different sites of the binding protein, thus explaining the effects of MyBP-C on the critical concentration of myosin polymerization.
Miyamoto
C A
CA
Instituto de Química, Universidade de São Paulo, Brazil.
Fischman
D A
DA
Reinach
F C
FC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D009218:Myosins; D000099163:Myosin Binding Protein C