Tropomodulin-binding site mapped to residues 7-14 at the N-terminal heptad repeats of tropomyosin isoform 5.
Tropomodulin is a globular protein that caps the pointed end of actin filaments by complexing with the N-terminus of a tropomyosin (TM) molecule. TM consists of coiled coils except for the N-terminus, which may be globular. Here we report that human TM isoform 5 (hTM5) lacking the N-terminal 18 residues lost its binding activity toward tropomodulin. We further characterized the tropomodulin-binding site by creating a series of deletion and missense mutations within this region, followed by a solid-phase binding assay. I7, V10, and I14, hydrophobic residues located at the a and d positions of N-terminal heptad repeats involving intertwine, are essential for tropomodulin binding. R12, a positively charged residue at the f position, is also involved in recognition. In contrast, A2R and G3Y mutations, each creating a bulky N-terminus, did not alter the binding. In addition, rat TM5b, which differs from hTM5 in residues 4-6, exhibits a similar binding affinity. The tropomodulin-binding site, therefore, is mapped to residues 7-14 at the beginning of the long heptad repeats. Column chromatography revealed that hTM5 mutants remained capable of dimerization. Results also suggest tropomodulin 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 binding. Since the N-terminal residues need to overlap with the C-terminus of TM in their head-to-tail association, this investigation elucidates the mechanisms by which the tropomodulin-hTM5 complex is formed and functions in regulating the actin filaments.
Vera
C
C
Department of Bioengineering and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093, USA.
Sood
A
A
Gao
K M
KM
Yee
L J
LJ
Lin
J J
JJ
Sung
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008840:Microfilament Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D051348:Tropomodulin; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D017931:DNA Primers; D008840:Microfilament Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C495829:TMOD1 protein, human; C498783:TPM3 protein, human; C495832:Tmod1 protein, rat; C498784:Tpm3 protein, rat; D051348:Tropomodulin; D014335:Tropomyosin