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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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Structure of “linker βS7–S8” from Cap32. Ribbon representation of the region around “linker βS7–S8” of the superposed Cap32 and chicken CapZβ structures. The residues of β-strands S7 and S8 of Cap32 and the two lysines K142 and K143 of chicken CapZ are shown as stick models. The part of Cap32 that is not visible in the electron density has been drawn illustrating the hypothetical positions of the lysines K142 and K143 of Dictyostelium Cap32 for comparison.
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Figure 6: Structure of “linker βS7–S8” from Cap32. Ribbon representation of the region around “linker βS7–S8” of the superposed Cap32 and chicken CapZβ structures. The residues of β-strands S7 and S8 of Cap32 and the two lysines K142 and K143 of chicken CapZ are shown as stick models. The part of Cap32 that is not visible in the electron density has been drawn illustrating the hypothetical positions of the lysines K142 and K143 of Dictyostelium Cap32 for comparison.

Mentions: In addition to the region connecting the β-strands of the central β-sheet of the α-subunits opposite to the β-tentacle, the crystal structure of Cap32/34 reveals a notable difference between the β-subunits of the two homologs. Due to disorder no electron density could be assigned to residues Gln-140–Gln-145 of the Cap32 central β-sheet (Figure 6). This region corresponds to a solvent-accessible turn region between S7 and S8 (corresponding to S6 and S7 in chicken Capβ), thus being referred to in this study as “linker βS7–S8” (β denotes the CP β-subunit). Since this segment is well-ordered in the CapZ structure, one could assume that the difference in flexibility might arise from “linker βS7–S8” undergoing conformational dynamics and serving as a binding site in the Cap32 molecule. As can be seen from the sequence alignment (Figure 5), “linker βS7–S8” harbours up to two basic amino acids (Lys-142, Lys-143 both in Cap32 from Dictyostelium discoideum and chicken Cap2, respectively), which, based on the crystal structure of CapZ, are positioned directly at the tip of the loop. Since acidic residues are not located in immediate proximity, the molecular surface of this region exhibits a pronounced positive electrostatic potential, making it particularly suitable for electrostatic interactions with negatively charged target sites. The sequence alignment further reveals that Lys-142 and Lys-143 of Cap32 are C-terminally flanked by three additional residues (Gly-144, Gln-145, Pro-146) resulting in an elongated linker region.


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)

Structure of “linker βS7–S8” from Cap32. Ribbon representation of the region around “linker βS7–S8” of the superposed Cap32 and chicken CapZβ structures. The residues of β-strands S7 and S8 of Cap32 and the two lysines K142 and K143 of chicken CapZ are shown as stick models. The part of Cap32 that is not visible in the electron density has been drawn illustrating the hypothetical positions of the lysines K142 and K143 of Dictyostelium Cap32 for comparison.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472329&req=5

Figure 6: Structure of “linker βS7–S8” from Cap32. Ribbon representation of the region around “linker βS7–S8” of the superposed Cap32 and chicken CapZβ structures. The residues of β-strands S7 and S8 of Cap32 and the two lysines K142 and K143 of chicken CapZ are shown as stick models. The part of Cap32 that is not visible in the electron density has been drawn illustrating the hypothetical positions of the lysines K142 and K143 of Dictyostelium Cap32 for comparison.
Mentions: In addition to the region connecting the β-strands of the central β-sheet of the α-subunits opposite to the β-tentacle, the crystal structure of Cap32/34 reveals a notable difference between the β-subunits of the two homologs. Due to disorder no electron density could be assigned to residues Gln-140–Gln-145 of the Cap32 central β-sheet (Figure 6). This region corresponds to a solvent-accessible turn region between S7 and S8 (corresponding to S6 and S7 in chicken Capβ), thus being referred to in this study as “linker βS7–S8” (β denotes the CP β-subunit). Since this segment is well-ordered in the CapZ structure, one could assume that the difference in flexibility might arise from “linker βS7–S8” undergoing conformational dynamics and serving as a binding site in the Cap32 molecule. As can be seen from the sequence alignment (Figure 5), “linker βS7–S8” harbours up to two basic amino acids (Lys-142, Lys-143 both in Cap32 from Dictyostelium discoideum and chicken Cap2, respectively), which, based on the crystal structure of CapZ, are positioned directly at the tip of the loop. Since acidic residues are not located in immediate proximity, the molecular surface of this region exhibits a pronounced positive electrostatic potential, making it particularly suitable for electrostatic interactions with negatively charged target sites. The sequence alignment further reveals that Lys-142 and Lys-143 of Cap32 are C-terminally flanked by three additional residues (Gly-144, Gln-145, Pro-146) resulting in an elongated linker region.

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