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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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Putative binding site for Z-disc proteins.A) Coil representation of Cap32/34 and chicken CapZ illustrating the B-factor distribution of the Cα atoms. B) Surface presentation of chicken CapZ with structural motifs defined and coloured as in Figure 1. Residues that are different between the α-subunit isoforms Capα1 and Capα2 are highlighted in black. These residues cluster in the same part of the α-globule that shows increased B-factors in the chicken CapZ structure.
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Figure 7: Putative binding site for Z-disc proteins.A) Coil representation of Cap32/34 and chicken CapZ illustrating the B-factor distribution of the Cα atoms. B) Surface presentation of chicken CapZ with structural motifs defined and coloured as in Figure 1. Residues that are different between the α-subunit isoforms Capα1 and Capα2 are highlighted in black. These residues cluster in the same part of the α-globule that shows increased B-factors in the chicken CapZ structure.

Mentions: Another difference between Cap32/34 and CapZ became apparent when the distribution of B-factor values was compared. As illustrated in Figure 7, the CapZ α- subunit segment spanning from Leu-101–Leu-117 exhibits a substantially higher average B-factor compared to the corresponding region in Cap32/34 (~86.8 Å2 for CapZ compared to ~24.2 Å2 for Cap32/34). Furthermore, the two homologs do not only adopt markedly different conformations within this part of the molecule (Cα r.m.s.d. of ~3.0 Å) but also display different secondary structural elements (residues Lys-103 – Pro-108 of CapZ exhibit a random coil structure, whereas the equivalent region in Cap32/34 is part of a β-strand). CapZ has recently been found to associate with the giant sarcomeric protein nebulin, which is thought to target the protein to the Z-disc [56]. Surprisingly, cytoplasmic CP also binds to nebulin in vitro, whereas in myocytes, which contain both CP variants, only CapZ has been found at the Z-disc [56]. Consequently, another binding partner might be responsible for targeting CapZ to the Z-disc. Based on our observation that the CapZ molecule includes a solvent-accessible region greatly differing in both flexibility and conformation from that of Cap32/34, residues Leu-101 – Leu-117 within the α-subunit may contribute to the interaction with the Z-disc of the sarcomere, either in an indirect manner by being involved in mediating the process or through direct binding. These conclusions are in line with the conformational flexibility of the neighbouring regions of the loops connecting S6 and S7, and S8 and S9 (see above) that also revealed differences between cytoplasmic CP and CapZ.


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)

Putative binding site for Z-disc proteins.A) Coil representation of Cap32/34 and chicken CapZ illustrating the B-factor distribution of the Cα atoms. B) Surface presentation of chicken CapZ with structural motifs defined and coloured as in Figure 1. Residues that are different between the α-subunit isoforms Capα1 and Capα2 are highlighted in black. These residues cluster in the same part of the α-globule that shows increased B-factors in the chicken CapZ structure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Putative binding site for Z-disc proteins.A) Coil representation of Cap32/34 and chicken CapZ illustrating the B-factor distribution of the Cα atoms. B) Surface presentation of chicken CapZ with structural motifs defined and coloured as in Figure 1. Residues that are different between the α-subunit isoforms Capα1 and Capα2 are highlighted in black. These residues cluster in the same part of the α-globule that shows increased B-factors in the chicken CapZ structure.
Mentions: Another difference between Cap32/34 and CapZ became apparent when the distribution of B-factor values was compared. As illustrated in Figure 7, the CapZ α- subunit segment spanning from Leu-101–Leu-117 exhibits a substantially higher average B-factor compared to the corresponding region in Cap32/34 (~86.8 Å2 for CapZ compared to ~24.2 Å2 for Cap32/34). Furthermore, the two homologs do not only adopt markedly different conformations within this part of the molecule (Cα r.m.s.d. of ~3.0 Å) but also display different secondary structural elements (residues Lys-103 – Pro-108 of CapZ exhibit a random coil structure, whereas the equivalent region in Cap32/34 is part of a β-strand). CapZ has recently been found to associate with the giant sarcomeric protein nebulin, which is thought to target the protein to the Z-disc [56]. Surprisingly, cytoplasmic CP also binds to nebulin in vitro, whereas in myocytes, which contain both CP variants, only CapZ has been found at the Z-disc [56]. Consequently, another binding partner might be responsible for targeting CapZ to the Z-disc. Based on our observation that the CapZ molecule includes a solvent-accessible region greatly differing in both flexibility and conformation from that of Cap32/34, residues Leu-101 – Leu-117 within the α-subunit may contribute to the interaction with the Z-disc of the sarcomere, either in an indirect manner by being involved in mediating the process or through direct binding. These conclusions are in line with the conformational flexibility of the neighbouring regions of the loops connecting S6 and S7, and S8 and S9 (see above) that also revealed differences between cytoplasmic CP and CapZ.

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