Cold-sensitive Mutants G680V and G691C ofDictyostelium Myosin II Confer Dramatically Different Biochemical Defects

Abstract

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.The study of the family of molecular motors recently has been invigorated by the availability of crystal structures of both the myosin (1-5) and kinesin family members (6, 7). This information, combined with a wealth of biochemical characterization of these motors, provides a platform on which to build a complete understanding of the mechanics of these pivotal cellular components. In a complementary approach, we have been generating conditionally functional (cold-sensitive) mutants of myosin II in the Dictyostelium system (8, 9). In our isolation of cold-sensitive myosin mutants, we uncovered two mutations in a biochemically renowned region of the protein, a helical element that houses two highly reactive cysteines in rabbit myosin.This region of myosin was first characterized because of the chemical accessibility of two cysteines contained within it (positions 697 (SH2) and 707 (SH1) in rabbit myosin). The crystal structure of chicken pectoralis muscle S1 displays these cysteines in a bent ␣-helix, separated from one another by 18 Å and facing opposite sides of the molecule (2). However, crosslinking experiments using rabbit skeletal muscle myosin demonstrate that during the stroking cycle, the two approach more closely than in the absence of nucleotide (10) and can be directly joined by a disulfide linkage, indicative of a separation of only about 3 Å and a substantially altered geometry (11). The conformational changes are not purely local, in that either cysteine can cross-link with residues in other parts of the structure (for example, SH1-Cys 522 , and SH2-Cys 540 in rabbit (12, 13)) during the cycle. Chemical modification studies have demonstrated that addition of bulky hydrophobic groups to the cysteines dramatically changes the properties of the myosin molecule, particularly with regard to the promiscuity and activity of its ATPase. The physiological substrate of myosin is Mg 2ϩ -ATP which supports only low-level ATPase activity in the absence of actin. However, if provided with Ca 2...

An actin–myosin-II interaction is involved in maintaining the contractile ring in fission yeast

“…3D,E). The G680V mutant myosin-II of Dictyostelium discoideum strongly binds to actin even in the presence of ATP by possibly forming a stable actin-myosin-II complex carrying ADP and phosphate (Patterson et al, 1997;Uyeda et al, 2002). The bulk of the corresponding Myo3-G688V mutant formed an aggregate in the medial region, whereas the rest localized faintly to the contractile ring (Fig.…”

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

“…The conserved glycine residue likely plays an essential role in the conformational changes around the actin-and nucleotide-binding sites, maybe functioning as a pivot point for the lever arm. Mutation of this glycine into an alanine residue dramatically alters the motor activity of the skeletal muscle myosin and the Dictyostelium discoideum myosin II, inhibiting the velocity of actin filament movement by 100-fold (Kinose et al, 1996;Patterson and Spudich, 1996;Patterson et al, 1997). Finally, class XIV myosins do not carry classical IQ motifs for the binding of light chains but harbor a conserved stretch of amino acids that might represent a very divergent form of this motif.…”

Mammalian Myosin-18A, a Highly Divergent Myosin

“…There is a conserved glycine (Gly 680 in the D. discoideum myosin-2 sequence) within the myosin superfamily that lies between the SH1 and SH2 helices and is thought to be in the vicinity of the rotation point of the lever arm. Mutation of this residue to amino acids other than glycine typically alters the nucleotide binding, hydrolysis, and release kinetics and dramatically lowers the in vitro motility (57). Recently, D. discoideum myosin-2 motor domain constructs bearing mutations to this residue were crystallized in different nucleotide states (58).…”