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

Abstract

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (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-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is ref lected 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-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (timeaveraged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar 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.The myosin superfamily comprises 15 classes of protein distinguished by the sequences of their catalytic domains but also differing in the domain organization of their tails (for reviews, see refs. 1-3). The two largest and best-studied classes are conventional (double-headed, filament-forming) class II myosins and unconventional (single-headed, monomeric) class I myosins. Class I was the first of the 14 classes of unconventional myosins to be discovered. The first known class I myosins, and arguably still the best biochemically described unconventional myosins of any class, are the myosins I of Acanthamoeba castellanii (4, 5).Each of the three AMI isozymes consists of a single, relatively short (for myosins) heavy chain and a single light chain (6). The heavy chain has an Ϸ72-kDa N-terminal catalytic domain that, as with all myosins, has an ATPase site and an ATP-sensitive actin-binding site. Importantly, a single Ser or Thr residue in their catalytic domains at a position occupied by a Glu or Asp in most other myosins (1) must be phosphorylated for full expression of their actin-activated ATPase activities (6, 7). Distal to the catalytic domain is a single 6-kDa IQ domain (the presumptive light chain-binding site) followed by a Ϸ50-kDa tail. The tail contains three distinctive regions: a basic, phospholipid-binding region; a Gly-Pro-Ala-rich (GPA) region that contains a second (ATPinsensitive) actin-binding site; and a src-homology 3 domain (SH3). The three regions are sequential in AMIB, but the SH3 domain splits the GPA region in AMIC (cf. Fig. 1).Similar myosins I occur in a wide variety of species, but there are also myosins I that lack the GPA and...

Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

“…If so, this difference may be revealed by cryoelectron microscopy of the complexes of F-actin with the constitutively active S329E and the constitutively inactive S329A mutants of Acanthamoeba myosin IC. The results of such a study are reported in the accompanying paper (47).…”

Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

“…The function of both myosins is to provide motile force through ATP hydrolysis within the myosin head domain. Myosin-I proteins are involved in cell locomotion, membrane transport, and phagocytosis, while myosin-II activity supports locomotion and the contractile ring that is formed during cell division (4,13).…”

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

“…Among their particularities, and contrary to the P. falciparum molecule, all the T. gondii myosins do not follow the TEDS rule (Heintzelman and Schwartzman, 1997; Figure 1A), which describes the presence of an acidic or phosphorylatable residue at a precise position close to the actin-binding region (originally mapped by Brzeska et al, 1989Brzeska et al, , 1990; for review, see Bement and Mooseker, 1995). In lower eukaryotes, phosphorylation of this conserved serine or threonine was shown to be crucial for the stimulation of the ATPase activity of class I myosins (Bement and Mooseker, 1995;Carragher et al, 1998;Novak and Titus, 1998). It is unclear at the moment whether and how these class XIV motors are activated and how conformational changes in the molecules occur.…”