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Conservation and divergence between cytoplasmic and muscle-specific actin capping proteins: insights from the crystal structure of cytoplasmic Cap32/34 from Dictyostelium discoideum.

Eckert C, Goretzki A, Faberova M, Kollmar M - BMC Struct. Biol. (2012)

Bottom Line: Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles.At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ).Significant structural flexibility could particularly be found within the α-subunit, a loop region in the β-subunit, and the surface of the α-globule where the amino acid differences between the cytoplasmic and sarcomeric mammalian CP are located.

View Article: PubMed Central - HTML - PubMed

Affiliation: Abteilung NMR basierte Strukturbiologie, Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077, Göttingen, Germany.

ABSTRACT

Background: Capping protein (CP), also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and β-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a "cap" by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles.

Results: To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32=β- and 34=α-subunit) from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle) and the flexible β-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the β-sheet region located opposite to the position of the C-terminal β-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the β-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ).

Conclusions: The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the α-subunit, a loop region in the β-subunit, and the surface of the α-globule where the amino acid differences between the cytoplasmic and sarcomeric mammalian CP are located. Hence, the crystal structure of Cap32/34 raises the possibility of different binding behaviours of the CP variants toward the barbed end of actin filaments, a feature, which might have arisen from adaptation to different environments.

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Crystal structure of Dictyostelium discoideum Cap32/34. A) Ribbon presentation of Cap32/34. The structural motifs are shown in different colours. For clarity and comparability we used the same motif and colour scheme as in [30]. The helices are numbered from the N- to the C-terminus. B) Top view of the structure highlighting the β-strands. Compared to CapZ, one more β-strand could be assigned to both the α-globule and the β-globule region. C) Superposition of Cap32 (red) and Cap34 (blue). While the globule regions are markedly similar, the N-stalk regions point to different directions demonstrating the pseudo 2-fold symmetry of the CP.
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Figure 1: Crystal structure of Dictyostelium discoideum Cap32/34. A) Ribbon presentation of Cap32/34. The structural motifs are shown in different colours. For clarity and comparability we used the same motif and colour scheme as in [30]. The helices are numbered from the N- to the C-terminus. B) Top view of the structure highlighting the β-strands. Compared to CapZ, one more β-strand could be assigned to both the α-globule and the β-globule region. C) Superposition of Cap32 (red) and Cap34 (blue). While the globule regions are markedly similar, the N-stalk regions point to different directions demonstrating the pseudo 2-fold symmetry of the CP.

Mentions: Crystals of the Cap32/34 protein were obtained by the hanging-drop vapour diffusion technique. The crystals belong to the tetragonal space group P41 with unit-cell parameters of a = 124.5, b = 124.5, c = 77.5 Å and α = β = γ = 90°, and contain two molecules per asymmetric unit (Table 1). The structure was solved by molecular replacement using the crystal structure of CapZ from Gallus gallus[32] as a search model (PDB code 1IZN). The structural model was refined to 2.2 Å resolution with a final Rwork of 22.6% and an Rfree of 26.5% (Figure 1). Superposition of the two Cap32/34 molecules within the asymmetric unit revealed only small deviations in their overall structures, with a root-mean-square deviation (r.m.s.d.) of 0.3 Å for 512 common Cα atoms. Equivalently to chicken CapZ [32], the α- and β-subunits of Cap32/34 from Dictyostelium discoideum have strikingly similar secondary and tertiary structures (Figure 1C), despite showing only modest homology at the amino acid sequence level. Furthermore, the two subunits are extensively intertwined, resulting in a pseudo 2-fold axis of rotational symmetry of the entire molecule. Given the tight interactions occurring between the CP subunits, it is not surprising that the heterodimer is extremely stable as opposed to the individual subunits. Briefly, Cap32/34 has the shape of a mushroom, comprising a stalk (“N-stalk”) and a cap (“central β-sheet” and “antiparallel H5s”). The mushroom stalk is composed of six anti-parallel α-helices, of which three are contributed from the N-terminus of each subunit (H1–3). Stretches of five antiparallel β-strands of the α-subunit (S1–5) and four of the β-subunit (S1–4) are located next to the stalk and under the cap of the mushroom (“α- and β-globule”). The cap consists of a single ten-stranded antiparallel β-sheet formed by five β-strands of each subunit (S6–10).


Conservation and divergence between cytoplasmic and muscle-specific actin capping proteins: insights from the crystal structure of cytoplasmic Cap32/34 from Dictyostelium discoideum.

Eckert C, Goretzki A, Faberova M, Kollmar M - BMC Struct. Biol. (2012)

Crystal structure of Dictyostelium discoideum Cap32/34. A) Ribbon presentation of Cap32/34. The structural motifs are shown in different colours. For clarity and comparability we used the same motif and colour scheme as in [30]. The helices are numbered from the N- to the C-terminus. B) Top view of the structure highlighting the β-strands. Compared to CapZ, one more β-strand could be assigned to both the α-globule and the β-globule region. C) Superposition of Cap32 (red) and Cap34 (blue). While the globule regions are markedly similar, the N-stalk regions point to different directions demonstrating the pseudo 2-fold symmetry of the CP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472329&req=5

Figure 1: Crystal structure of Dictyostelium discoideum Cap32/34. A) Ribbon presentation of Cap32/34. The structural motifs are shown in different colours. For clarity and comparability we used the same motif and colour scheme as in [30]. The helices are numbered from the N- to the C-terminus. B) Top view of the structure highlighting the β-strands. Compared to CapZ, one more β-strand could be assigned to both the α-globule and the β-globule region. C) Superposition of Cap32 (red) and Cap34 (blue). While the globule regions are markedly similar, the N-stalk regions point to different directions demonstrating the pseudo 2-fold symmetry of the CP.
Mentions: Crystals of the Cap32/34 protein were obtained by the hanging-drop vapour diffusion technique. The crystals belong to the tetragonal space group P41 with unit-cell parameters of a = 124.5, b = 124.5, c = 77.5 Å and α = β = γ = 90°, and contain two molecules per asymmetric unit (Table 1). The structure was solved by molecular replacement using the crystal structure of CapZ from Gallus gallus[32] as a search model (PDB code 1IZN). The structural model was refined to 2.2 Å resolution with a final Rwork of 22.6% and an Rfree of 26.5% (Figure 1). Superposition of the two Cap32/34 molecules within the asymmetric unit revealed only small deviations in their overall structures, with a root-mean-square deviation (r.m.s.d.) of 0.3 Å for 512 common Cα atoms. Equivalently to chicken CapZ [32], the α- and β-subunits of Cap32/34 from Dictyostelium discoideum have strikingly similar secondary and tertiary structures (Figure 1C), despite showing only modest homology at the amino acid sequence level. Furthermore, the two subunits are extensively intertwined, resulting in a pseudo 2-fold axis of rotational symmetry of the entire molecule. Given the tight interactions occurring between the CP subunits, it is not surprising that the heterodimer is extremely stable as opposed to the individual subunits. Briefly, Cap32/34 has the shape of a mushroom, comprising a stalk (“N-stalk”) and a cap (“central β-sheet” and “antiparallel H5s”). The mushroom stalk is composed of six anti-parallel α-helices, of which three are contributed from the N-terminus of each subunit (H1–3). Stretches of five antiparallel β-strands of the α-subunit (S1–5) and four of the β-subunit (S1–4) are located next to the stalk and under the cap of the mushroom (“α- and β-globule”). The cap consists of a single ten-stranded antiparallel β-sheet formed by five β-strands of each subunit (S6–10).

Bottom Line: Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles.At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ).Significant structural flexibility could particularly be found within the α-subunit, a loop region in the β-subunit, and the surface of the α-globule where the amino acid differences between the cytoplasmic and sarcomeric mammalian CP are located.

View Article: PubMed Central - HTML - PubMed

Affiliation: Abteilung NMR basierte Strukturbiologie, Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077, Göttingen, Germany.

ABSTRACT

Background: Capping protein (CP), also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and β-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a "cap" by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles.

Results: To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32=β- and 34=α-subunit) from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle) and the flexible β-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the β-sheet region located opposite to the position of the C-terminal β-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the β-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ).

Conclusions: The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the α-subunit, a loop region in the β-subunit, and the surface of the α-globule where the amino acid differences between the cytoplasmic and sarcomeric mammalian CP are located. Hence, the crystal structure of Cap32/34 raises the possibility of different binding behaviours of the CP variants toward the barbed end of actin filaments, a feature, which might have arisen from adaptation to different environments.

Show MeSH
Related in: MedlinePlus