Kinetic Characterization of Converter and Relay Loop Domain Interaction in Drosophila Myosin Sub-Fragment 1

Abstract

We investigated the kinetic properties of Drosophila myosin sub-fragment 1 (S-1) mutant R759E and its suppressor R759E/N509K. The R759E mutation is located in the converter domain of the myosin head whereas N509K is present at the relay loop. Steady-state measurements for R759E S-1 show more than 50% reduction in calcium ATPase as well as ∼40 % reduction in basal magnesium ATPase compared to wild type whereas actin-stimulated Mg-ATPase of R759E (Vmax) is reduced ∼70 %. Homology models of myosin S-1 suggest the R759E mutation can disrupt the interface between the converter and the relay loop and that the suppressor (R759E/N509K) can restore this interaction. This prediction was confirmed by our steady-state ATPase data, which demonstrated that the suppressor mutation restored calcium, basal Mg-ATPase and Vmax back towards wild type values. Calcium and basal Mg-ATPase activity of R759E/N509K increased ∼30 % and ∼25 % respectively compared to mutant R759E S-1. Actin-stimulated Mg-ATPase activity of R759E/N509K was ∼40 % higher compared to R759E mutant S-1. Using flash photolysis, our transient kinetics results showed no significant change in the ATP-induced dissociation of acto-S-1 (K1k+2′) and ADP-affinity (KAD) of acto-S-1 for R759E and R759E/N509K compared to wild-type. Using stopped-flow we measured ATP-binding to S-1 (R759E and R759E/N509K). Compared to wild-type we find for R759E a 30% reduction in the rate constant of ATP-binding (K1k+2) and a 25% reduction in the rate constant of ATP-hydrolysis (k+3+k-3) whereas for R759E/N509K ATP-hydrolysis is restored to wild-type level. This demonstrates the significance of relay-loop and converter domain interaction for S-1's enzymatic ability. The ability to suppress many of the mutant defects in S-1 kinetics corroborates the dramatically improved muscle function we previously found in R759E/N509K double mutants and provides mechanistic insight into this improvement.