Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis.
Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.
MRC Laboratory of Molecular Biology, Cambridge, England.
E J
EJ
Mitchell
J
J
Karn
D M
DM
Brown
A
A
Newman
R
R
Jakes
J
J
Kendrick-Jones
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D015879:Myosin Subfragments; D009218:Myosins / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping
D015879:Myosin Subfragments; D010446:Peptide Fragments; D009218:Myosins