Functional diversity between orthologous myosins with minimal sequence diversity M. CANEPARI 1 , R. ROSSI 1 , M. A. PELLEGRINO 1 , R. BOTTINELLI 1 , S. SCHIAFFINO 2; and C. REGGIANI 3 1 Institute of Human Physiology, University of Pavia, Pavia; 2 Department of Biomedical Sciences and CNR Centre of Muscle Biology and Physiopathology, University of Padova, Padova; 3 Department of Anatomy and Physiology, University of Padova, Padova, Italy Received 10 March 2000; accepted in revised form 26 May 2000 Abstract To de®ne the structural dierences that are responsible for the functional diversity between orthologous sarcomeric myosins, we compared the rat and human b/slow myosins. Functional comparison showed that rat b/slow myosin has higher ATPase activity and moves actin ®laments at higher speed in in vitro motility assay than human b/slow myosin. Sequence analysis shows that the loop regions at the junctions of the 25 and 50 kDa domains (loop 1) and the 50 and 20 kDa domains (loop 2), which have been implicated in determining functional diversity of myosin heavy chains, are essentially identical in the two orthologs. There are only 14 non-conservative substitutions in the two myosin heavy chains, three of which are located in the secondary actin-binding loop and ¯anking regions and others correspond to residues so far not assigned a functional role, including two residues in the proximal S2 domain. Interestingly, in some of these positions the rat b/slow myosin heavy chain has the same residues found in human cardiac a myosin, a fast-type myosin, and fast skeletal myosins. These observations indicate that functional and structural analysis of myosin orthologs with limited sequence diversity can provide useful clues to identify amino acid residues involved in modulating myosin function. Introduction Myosin is a molecular motor that moves actin ®laments by converting the chemical energy of ATP into mechan- ical energy. The chemo-mechanical transduction is per- formed at dierent rates by various myosin isoforms. The molecular determinants of the functional diversity between myosin isoforms still await to be completely identi®ed. Myosin heavy chain (MyHC), the subunit in which enzymatic and motor function are localized, is a very conserved molecule. Some sequences in the actin- binding surface, the catalytic site and the converter region are virtually identical in all myosin isoforms. Variable regions include two ¯exible loops located at the surface of the myosin head, which are particularly sensitive to proteolytic cleavage. A ®rst loop connects the 25 kDa and the 50 kDa domain of MyHC (loop 1, located near the catalytic site), whereas a second loop connects the 50 kDa and the 20 kDa domain (loop 2, located at the actin interface). It has been proposed that the amino acid sequences of these loops are responsible for the diversity in actin displacement rate and ATP hydrolysis rate, respectively, among myosin isoforms (Spudich, 1994). The composition of these loops is known to vary among MyHC isoforms and their length, charge and presence of proline residues can aect ATPase activity and/or in vitro motility [see for a recent review (Geeves and Holmes, 1999)]. The ®nding that the sequences of the loops are evolutionary more conserved when orthologous myosins, which are expected to be kinetically similar, are com- pared, has also been taken as evidence that the loops are involved in determining specialized functional character- istics (Goodson et al., 1999). One approach used to explore the role of the loops has been to generate chimaeric myosins by transplant- ing selected sequences of dierent donor myosins into the same acceptor myosin molecule. The replacement of the sequence of loop 2 in Dictyostelium myosin with corresponding sequences of other myosins generates chimaeric myosins whose ATPase rate, but not speed of actin translation, resembles that of the donor myosins (Uyeda et al., 1994). However, the insertion of loop 1 sequences of several myosin isoforms into a smooth muscle myosin generate chimaeric myosins whose speed of actin translation and ADP release rate are not correlated with those of the donor myosins (Sweeney et al., 1998). So far, the `chimaeric myosin' approach has been widely applied only to smooth muscle and Dictyostelium myosins and not to sarco- meric myosins. An alternative approach to identify the critical resi- dues in the myosin head which regulate ATPase activity and speed of movement is to compare MyHC sequence in natural variants of myosin showing dierent func- tional properties [see Geeves and Holmes (1999) and *To whom correspondence should be addressed: Tel.: +39 49 827 6034; Fax: +39 49 827 6040; E-mail: schia@civ.bio.unipd.it Journal of Muscle Research and Cell Motility 21: 375±382, 2000. 375 Ó 2000 Kluwer Academic Publishers. Printed in the Netherlands.