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A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Liang W, Warrick HM, Spudich JA - J. Cell Biol. (1999)

Bottom Line: Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes.These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments.The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.

ABSTRACT
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes. Here we report a suppressor screen of Dictyostelium myosin II- cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

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A model for the bent conformation responsible for the regulation of the initial step of filament assembly. We propose that regulation of the initial step of filament assembly results from the state of phosphorylation in the myosin II molecules. An unphosphorylated myosin II molecule in the straight conformation is shown below. The two regions (A and B) rich in alanines are shown in dashed patterns. Mutations mapped from the suppressors against 3×Asp myosins are shown in orange (for single point mutations) and green (for deletion mutations), and locate in region B. A bent phosphorylated myosin II molecule that folds at ∼1,200 Å from the head–neck junction is shown, where the alanine-rich regions A and B overlap. Threonine pair 1823/1833 may be a nucleation site where the bent monomer conformation is initiated after phosphorylation, followed by the formation of an antiparallel tetrameric coiled–coil in the alanine-rich regions. The assembly domain (O'Halloran et al. 1990; Shoffner and De Lozanne 1996; shown in blue) is sequestered as a looped structure that is unable to self-assemble into higher-order structures.
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Figure 7: A model for the bent conformation responsible for the regulation of the initial step of filament assembly. We propose that regulation of the initial step of filament assembly results from the state of phosphorylation in the myosin II molecules. An unphosphorylated myosin II molecule in the straight conformation is shown below. The two regions (A and B) rich in alanines are shown in dashed patterns. Mutations mapped from the suppressors against 3×Asp myosins are shown in orange (for single point mutations) and green (for deletion mutations), and locate in region B. A bent phosphorylated myosin II molecule that folds at ∼1,200 Å from the head–neck junction is shown, where the alanine-rich regions A and B overlap. Threonine pair 1823/1833 may be a nucleation site where the bent monomer conformation is initiated after phosphorylation, followed by the formation of an antiparallel tetrameric coiled–coil in the alanine-rich regions. The assembly domain (O'Halloran et al. 1990; Shoffner and De Lozanne 1996; shown in blue) is sequestered as a looped structure that is unable to self-assemble into higher-order structures.

Mentions: The results presented here, together with earlier results (Pasternak et al. 1989; Tan et al. 1992), suggest the following structural model of phosphorylation control of myosin II thick filament assembly (Fig. 7). Phosphorylation by myosin II heavy chain kinase produces charges on the outside of the coiled–coil tail that help stabilize the bent form of the myosin. Bent myosin II molecules cannot associate with other molecules to form parallel dimers, and therefore no antiparallel tetramers appear for the next phase of filament formation. Myosin II heavy chain phosphatase removes phosphates from the bent monomers, and the molecules return to their straight conformation. We propose that the threonine pair 1823/1833 could act as a nucleation site, which when phosphorylated, initiates the bent monomer conformation by orienting the two strands of dimeric coiled–coils. Once nucleated, regions A and B may zip up into an antiparallel four-stranded structure, possibly similar to that observed for the ColE1 Rop protein (Banner et al. 1987). Formation of such a structure results in a major bend at ∼1,200 Å from the head–neck junction. Moreover, the previously identified assembly domain (Fig. 7, blue) (O'Halloran and Spudich 1990) is sequestered as a loop between regions A and B. This conformation of the assembly domain prevents intermolecular interactions that lead to formation of thick filaments. The equilibrium between the bent versus straight conformations is delicately poised, and can be easily disturbed by mutations at multiple sites in the tail (e.g., the suppressor mutations described here; see also Moores and Spudich 1998).


A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Liang W, Warrick HM, Spudich JA - J. Cell Biol. (1999)

A model for the bent conformation responsible for the regulation of the initial step of filament assembly. We propose that regulation of the initial step of filament assembly results from the state of phosphorylation in the myosin II molecules. An unphosphorylated myosin II molecule in the straight conformation is shown below. The two regions (A and B) rich in alanines are shown in dashed patterns. Mutations mapped from the suppressors against 3×Asp myosins are shown in orange (for single point mutations) and green (for deletion mutations), and locate in region B. A bent phosphorylated myosin II molecule that folds at ∼1,200 Å from the head–neck junction is shown, where the alanine-rich regions A and B overlap. Threonine pair 1823/1833 may be a nucleation site where the bent monomer conformation is initiated after phosphorylation, followed by the formation of an antiparallel tetrameric coiled–coil in the alanine-rich regions. The assembly domain (O'Halloran et al. 1990; Shoffner and De Lozanne 1996; shown in blue) is sequestered as a looped structure that is unable to self-assemble into higher-order structures.
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Related In: Results  -  Collection

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Figure 7: A model for the bent conformation responsible for the regulation of the initial step of filament assembly. We propose that regulation of the initial step of filament assembly results from the state of phosphorylation in the myosin II molecules. An unphosphorylated myosin II molecule in the straight conformation is shown below. The two regions (A and B) rich in alanines are shown in dashed patterns. Mutations mapped from the suppressors against 3×Asp myosins are shown in orange (for single point mutations) and green (for deletion mutations), and locate in region B. A bent phosphorylated myosin II molecule that folds at ∼1,200 Å from the head–neck junction is shown, where the alanine-rich regions A and B overlap. Threonine pair 1823/1833 may be a nucleation site where the bent monomer conformation is initiated after phosphorylation, followed by the formation of an antiparallel tetrameric coiled–coil in the alanine-rich regions. The assembly domain (O'Halloran et al. 1990; Shoffner and De Lozanne 1996; shown in blue) is sequestered as a looped structure that is unable to self-assemble into higher-order structures.
Mentions: The results presented here, together with earlier results (Pasternak et al. 1989; Tan et al. 1992), suggest the following structural model of phosphorylation control of myosin II thick filament assembly (Fig. 7). Phosphorylation by myosin II heavy chain kinase produces charges on the outside of the coiled–coil tail that help stabilize the bent form of the myosin. Bent myosin II molecules cannot associate with other molecules to form parallel dimers, and therefore no antiparallel tetramers appear for the next phase of filament formation. Myosin II heavy chain phosphatase removes phosphates from the bent monomers, and the molecules return to their straight conformation. We propose that the threonine pair 1823/1833 could act as a nucleation site, which when phosphorylated, initiates the bent monomer conformation by orienting the two strands of dimeric coiled–coils. Once nucleated, regions A and B may zip up into an antiparallel four-stranded structure, possibly similar to that observed for the ColE1 Rop protein (Banner et al. 1987). Formation of such a structure results in a major bend at ∼1,200 Å from the head–neck junction. Moreover, the previously identified assembly domain (Fig. 7, blue) (O'Halloran and Spudich 1990) is sequestered as a loop between regions A and B. This conformation of the assembly domain prevents intermolecular interactions that lead to formation of thick filaments. The equilibrium between the bent versus straight conformations is delicately poised, and can be easily disturbed by mutations at multiple sites in the tail (e.g., the suppressor mutations described here; see also Moores and Spudich 1998).

Bottom Line: Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes.These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments.The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.

ABSTRACT
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II- phenotypes. Here we report a suppressor screen of Dictyostelium myosin II- cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

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