Sequence variations in the surface loop near the nucleotide binding site modulate the ATP turnover rates of molluscan myosins

Abstract

The muscle and species-specific differences in enzymatic activity between Placopecten and Argopecten striated and catch muscle myosins are attributable to the myosin heavy chain. To identify sequences that may modulate these differences, we cloned and sequenced the cDNA encoding the myosin heavy chains of Placopecten striated and catch muscle. Deduced protein sequences indicate two similar isoforms in catch and striated myosins (97% identical); variations arise by differential RNA splicing of five alternative exons from a single myosin heavy chain gene. The first encodes the phosphate-binding loop; the second, part of the ATP binding site; the third, part of the actin binding site; the fourth, the hinge in the rod; and the fifth, a tailpiece found only in the catch muscle myosin heavy chain. Both Placopecten myosin heavy chains are 96% identical to Argopecten myosin heavy chaina isoforms. Because subfragment-1 ATPase activities reflect the differences observed in the parent myosins, the motor domain is responsible for the variations in ATPase activities. In addition, data show that differences are due to Vmax and not actin affinity. The sequences of all four myosin heavy chain motor domains diverge only in the flexible surface loop near the nucleotide binding pocket. Thus, the different ATPase activities of four molluscan muscle myosins are likely due to myosin heavy chain sequence variations within the flexible surface loop that forms part of the ATP binding pocket of the motor domain.

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

“…We have formed an LCD which mimics that of Placopecten catch muscle with 100% RLC SmoA. The Placopecten striated muscle was used to obtain the ELC and the heavy chain fragment (HCF) for several reasons: it is much more plentiful than the catch muscle, gives a fourfold myosin yield over catch muscle, and the amino acid sequences of the HCF portion and the ELC of the striated muscle are identical to those of the catch muscle (21,43).…”

The myosin power stroke

“…However, this type of segregation may not be a universal feature of all myosin isoforms. For example, data from various Loop 1 [Kurzawa-Goertz et al, 1998;Perreault-Micale et al, 1996] and Loop 2 chimera [Rovner, 1998] in myosin backbones other than Dictyostelium myosin-II lead to a more complex story, where effects on actin-activated ATPase rates and actin filament sliding velocities cannot be attributed to a specific loop. However, an interesting relationship was determined for Loop 1 chimera in expressed smooth muscle HMM ].…”

Locking Regulatory Myosin in the Off-state with Trifluoperazine

“…With respect to the chicken smooth muscle myosin structure (35), the G-helix within the TFP-binding lobe of E-LC abuts the motor domain close to the 25/50-kDa loop (Ala 198 -Lys 204 ) (Fig. 7c), a region of the heavy chain implicated as a major determinant in the rate of ADP release (37)(38)(39)(40)(41) (Fig. 7d) within the so-called SH3 domain, which has been suggested to limit the potential swing of the lever arm (35).…”

“…We speculate that the interface between the 25/50-kDa loop and E-LC is most likely to participate in the off-state (as seen in Fig. 7c) because the 25/50-kDa loop is known to be a major determinant of the rate of ADP release (37)(38)(39)(40)(41), and the rate-limiting state in the absence of calcium is likely to be a weak binding ADP⅐P i intermediate (45,46), not an ATP-like structure. Further work is needed to test this hypothesis through both structural and functional means.…”