Structural invariance of constitutively active and inactive mutants of Acanthamoeba myosin IC bound to F-actin in the rigor and ADP-bound states

The three single-headed monomeric myosin I isozymes of Acanthamoeba castell anii (AMIs)-AMIA, AMIB, and AMIC-are among the best-studied of all myosins, We have used AMIC to study structural correlates of myosin's actin-activat ed ATPase, This activity is normally controlled by phosphorylation of Ser-3 29, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively, To determine wh ether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-A ngstrom resolution by using cryoelectron microscopy and image reconstructio n. No such difference was observed. Consequently, we suggest that regulatio n may be affected not by altering the static (time-averaged) structure of A MIC but by modulating its dynamic properties, i.e,, molecular breathing. Th e tail domain of vertebrate intestinal brush-border myosin I has been obser ved to swing through 31 degrees on binding of ADP, However, it was predicte d on grounds of differing kinetics that any such effects with AMIC should b e small [Jontes, J. D., Ostap, E, M,, Pollard, T, D, & Milligan, R, A. (199 8) J, Cell Biol. 141, 155-162], We have confirmed this hypothesis by observ ing actin-associated AMIC in its ADP-bound state, Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under sim ilar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.