title: The N-terminus of A1-type myosin essential light chains binds actin and modulates myosin motor function.
abstract: There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1 (S1), S1(A1) and S1(A2). The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent Km for actin and a slower turnover of MgATP (k(cat)) than S1(A2). The two forms of the ELC differ only at their N-termini where A1 has an additional 40-odd amino acids that are not present in A2. The human atrial ELC (an A1-type ELC) was overexpressed in Escherichia coli and purified by ammonium sulphate fractionation and ion-exchange chromatography. The recombinant ELC had actin-activated MgATPase kinetics similar to those for rabbit skeletal S1(A1) under the same conditions. Deletion of the first 45 amino acid residues resulted in an ELC similar to the rabbit skeletal A2 isoform and, when hybridised into S1, in S1(A2)-like kinetic properties. Results obtained with an ELC mutant that lacks the first 11 residues were intermediate between these two extremes but tending towards the S1(A2)-like phenotype. The wild-type ELC (both hybridised into S1 or free in solution) could be cross-linked to F-actin, whereas the deletion mutant lacking the first 45 amino acids could not. The deletion mutant lacking the first 11 amino acids cross-linked only poorly under the same conditions, consistent with the MgATPase data. We therefore conclude that these N-terminal eleven amino acids predominantly encode an actin-binding site which modulates the kinetics of the myosin motor. Furthermore, while free A1-type ELC cross-linked to both polymeric F-actin and the monomeric G-actin:DNase-I complex, the same ELC in S1(A1) could only cross-link to F-actin. This suggests that the light chain binds to a different actin monomer than the heavy chain.
authors.affiliation: School of Biochemistry, The University of Birmingham, United Kingdom.
authors.forename: D J
authors.initials: DJ
authors.lastname: Timson
authors.forename: H R
authors.initials: HR
authors.lastname: Trayer
authors.forename: I P
authors.initials: IP
authors.lastname: Trayer
mesh_terms: D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry* / Q000502:physiology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000199:Actins; D007527:Isoenzymes; D018994:Myosin Light Chains; D011994:Recombinant Proteins; D017301:Ca(2+) Mg(2+)-ATPase