Are there two different binding sites for ATP on the myosin head, or only one that switches between two conformers?

Striated muscle contraction depends on the cyclic interaction of the motor domains of myosin 2 (“heads” or S1) with actin, tightly coupled to ATP hydrolysis. It is generally thought that each myosin head contains an actin and an ATP binding site whose cross-talk gives rise to the cross-bridge cycle (Geeves and Holmes 2005; Sweeney and Houdusse 2010). In the last 30 years, kinetic and structural studies greatly improved the understanding of the relation between the states of the actomyosin ATPase cycle and mechanical activity, showing highly conserved features in the myosin superfamily. However, despite this consensus, there is uncertainty as to the number of ATP sites on the myosin head (for instance Tesi et al. 1989). In a recent study, Brenner and co-workers proposed that each of the two myosin heads has only one site for ATP switching between two conformers (Amrute-Nayak et al. 2014). They investigated the ATPase kinetics of individual myosin molecules by the use of fluorescently labelled ATP and measuring “residence times” of the labelled ATP on individual active sites from “dwell” and “waiting” times of the fluorescent signals. They found that the termination of fluorescence could originate from two different pathways as two different dwell time populations were identified in all the myosins tested (including rabbit psoas): a “long” lived time state consistent with conventional ATPase measurements from solution studies and a “short” lived time state. From kinetic modelling studies Amrute-Nayak et al. (2014) proposed that the site for ATP on the myosin head can switch between two conformers—one allowing the complete ATPase cycle, which they labelled the M conformer, another site that binds ATP reversibly, the M′ conformer. The intriguing proposal of Brenner and co-workers induced us to resurrect structural and transient kinetic data on the different myosin systems carried out in the 1980s from which we proposed that ATP interacts with different sites on the myosin head: an ATPase site and a site that is involved in the actomyosin dissociation.