Functional diversity between orthologous myosins with minimal sequence diversity

To define the structural differences that are responsible for the functiona l diversity between orthologous sarcomeric myosins, we compared the rat and human beta/slow myosins. Functional comparison showed that rat beta/slow m yosin has higher ATPase activity and moves actin filaments at higher speed in in vitro motility assay than human beta/slow myosin. Sequence analysis s hows that the loop regions at the junctions of the 25 and 50 kDa domains (l oop 1) and the 50 and 20 kDa domains (loop 2), which have been implicated i n determining functional diversity of myosin heavy chains, are essentially identical in the two orthologs. There are only 14 non-conservative substitu tions in the two myosin heavy chains, three of which are located in the sec ondary actin-binding loop and flanking regions and others correspond to res idues so far not assigned a functional role, including two residues in the proximal S2 domain. Interestingly, in some of these positions the rat beta/ slow myosin heavy chain has the same residues found in human cardiac alpha myosin, a fast-type myosin, and fast skeletal myosins. These observations i ndicate that functional and structural analysis of myosin orthologs with li mited sequence diversity can provide useful clues to identify amino acid re sidues involved in modulating myosin function.