Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly⁴⁷⁴ and Asn⁵⁰⁹ are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.