Tropomodulin-binding site mapped to residues 7-14 at the N-terminal heptadrepeats of tropomyosin isoform 5

Tropomodulin is a globular protein that caps the pointed end of actin filam ents by complexing with the N-terminus of a tropomyosin (TRI) molecule. TM consists of coiled coils except for the N-terminus, which may be globular. Here we report that human TRI isoform 5 (hTM5) lacking the N-terminal 18 re sidues lost its binding activity toward tropomodulin, We further characteri zed the tropomodulin-binding site by creating a series of deletion and miss ense mutations within this region, followed by a solid-phase binding assay. I-7, V-10, and I-14, hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropo modulin binding. R-12, a positively charged residue at the f position, is a lso involved in recognition. In contrast, A2R and G3Y mutations, each creat ing a bulky N-terminus, did not alter the binding. In addition, rat TM5b, w hich differs from hTM5 in residues 4-6, exhibits a similar binding affinity . The tropomodulin-binding site, therefore, is mapped to residues 7-14 at t he beginning of the long heptad repeats. Column chromatography revealed tha t hTM5 mutants remained capable of dimerization. Results also suggest tropo modulin has a groove-type, rather than a cavity-type, binding site for hTM5 . We also mapped the epitope of monoclonal antibody LC1 to residues 4-10 of hTM5 and showed the competition between mAb LC1 and tropomodulin in hTM5 b inding. Since the N-terminal residues need to overlap with the C-terminus o f TM in their head-to-tail association, this investigation elucidates the m echanisms by which the tropomodulin-hTM5 complex is formed and functions in regulating the actin filaments. (C) 2000 Academic Press.