2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1

► The compound 2,4-dinitrophenol (DNP) binds near a helix-loop-helix that includes a reactive lysine (Lys553) in myosin's lower 50-kDa subdomain, activating ATP hydrolysis. ► Reactivity of this lysine is reduced differentially by a broad range of ligands (nucleotide analogs, actin and DNP), indicating significant conformational flexibility among subdomains along the entire pathway from ATP pocket to the actin-binding region. ► Lys553 reactivity appears to be correlated with progressive closure of the 50-kDa cleft rather than changes in the status of switch 2. We suggest the DNP stabilizes the cleft in a partially open state.