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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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Sequence conservation within the actin-binding region of the β-subunits. The sequence logos are based on 299 β-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the β-subunits. Two regions known to be important for actin-binding are shown (For the representation of the entire β-subunits see Additional file 3). For better orientation, the sequences of three representative β-subunits are shown: chicken Cap2 of which all previous crystal structures have been obtained, the yeast Cap2 as one of the targets of mutagenesis experiments, and Dictyostelium Cap32 whose structure is presented here. Secondary structural elements, important residues indicating various interactions, and taxa/species with elongated loops are denoted as in Figure 4 (The full-length multiple sequence alignment of the β-subunits is available as Additional File 4). Loops, which exist only in single species, have been removed to shorten the alignment by the number of residues as indicated. Numbering below the logos refers to positions in the multiple sequence alignment.
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Figure 5: Sequence conservation within the actin-binding region of the β-subunits. The sequence logos are based on 299 β-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the β-subunits. Two regions known to be important for actin-binding are shown (For the representation of the entire β-subunits see Additional file 3). For better orientation, the sequences of three representative β-subunits are shown: chicken Cap2 of which all previous crystal structures have been obtained, the yeast Cap2 as one of the targets of mutagenesis experiments, and Dictyostelium Cap32 whose structure is presented here. Secondary structural elements, important residues indicating various interactions, and taxa/species with elongated loops are denoted as in Figure 4 (The full-length multiple sequence alignment of the β-subunits is available as Additional File 4). Loops, which exist only in single species, have been removed to shorten the alignment by the number of residues as indicated. Numbering below the logos refers to positions in the multiple sequence alignment.

Mentions: In contrast to the α-tentacle, neither Cap32/34 nor CapZ crystals grown at physiological pH provided an interpretable electron density for the C-terminal segment of the β-subunit (β-tentacle), indicating that this part of the CP molecule is highly mobile. Molecular dynamics studies confirmed the highly flexible nature of this region [53] and NMR experiments showed that the β-tentacle adopts a coil structure in solution [49]. Crystals of native CapZ have previously been soaked into an acidic solution, which stabilized the β-tentacle and allowed its structure to be solved [32]. Hereby it was demonstrated that the β-tentacle also comprises a short amphipathic α-helix, which, more importantly, extends out from the main body of the protein without making any specific interactions with CP. Although the β-tentacle sequence is not conserved in general, the three hydrophobic positions (residues L258, L262, and L266 in GgCapZ) at intervals of four residues are conserved (Figure 5) and exchanging them by polar residues abolishes actin-binding [50]. Therefore, CP has been proposed to bind to actin in two steps—first electrostatically through the basic patch on its α-subunit’s C-terminus, followed by hydrophobic interactions via its amphipathic β-tentacle [11]. The β-tentacles’ helical structure is stabilized in the crystal structure by interaction with a symmetry-related molecule [32]. We also soaked the Dictyostelium Cap32/34 crystals in acidic solution but did not see additional electron density in the region where the β-tentacle would be located.


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)

Sequence conservation within the actin-binding region of the β-subunits. The sequence logos are based on 299 β-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the β-subunits. Two regions known to be important for actin-binding are shown (For the representation of the entire β-subunits see Additional file 3). For better orientation, the sequences of three representative β-subunits are shown: chicken Cap2 of which all previous crystal structures have been obtained, the yeast Cap2 as one of the targets of mutagenesis experiments, and Dictyostelium Cap32 whose structure is presented here. Secondary structural elements, important residues indicating various interactions, and taxa/species with elongated loops are denoted as in Figure 4 (The full-length multiple sequence alignment of the β-subunits is available as Additional File 4). Loops, which exist only in single species, have been removed to shorten the alignment by the number of residues as indicated. Numbering below the logos refers to positions in the multiple sequence alignment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Sequence conservation within the actin-binding region of the β-subunits. The sequence logos are based on 299 β-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the β-subunits. Two regions known to be important for actin-binding are shown (For the representation of the entire β-subunits see Additional file 3). For better orientation, the sequences of three representative β-subunits are shown: chicken Cap2 of which all previous crystal structures have been obtained, the yeast Cap2 as one of the targets of mutagenesis experiments, and Dictyostelium Cap32 whose structure is presented here. Secondary structural elements, important residues indicating various interactions, and taxa/species with elongated loops are denoted as in Figure 4 (The full-length multiple sequence alignment of the β-subunits is available as Additional File 4). Loops, which exist only in single species, have been removed to shorten the alignment by the number of residues as indicated. Numbering below the logos refers to positions in the multiple sequence alignment.
Mentions: In contrast to the α-tentacle, neither Cap32/34 nor CapZ crystals grown at physiological pH provided an interpretable electron density for the C-terminal segment of the β-subunit (β-tentacle), indicating that this part of the CP molecule is highly mobile. Molecular dynamics studies confirmed the highly flexible nature of this region [53] and NMR experiments showed that the β-tentacle adopts a coil structure in solution [49]. Crystals of native CapZ have previously been soaked into an acidic solution, which stabilized the β-tentacle and allowed its structure to be solved [32]. Hereby it was demonstrated that the β-tentacle also comprises a short amphipathic α-helix, which, more importantly, extends out from the main body of the protein without making any specific interactions with CP. Although the β-tentacle sequence is not conserved in general, the three hydrophobic positions (residues L258, L262, and L266 in GgCapZ) at intervals of four residues are conserved (Figure 5) and exchanging them by polar residues abolishes actin-binding [50]. Therefore, CP has been proposed to bind to actin in two steps—first electrostatically through the basic patch on its α-subunit’s C-terminus, followed by hydrophobic interactions via its amphipathic β-tentacle [11]. The β-tentacles’ helical structure is stabilized in the crystal structure by interaction with a symmetry-related molecule [32]. We also soaked the Dictyostelium Cap32/34 crystals in acidic solution but did not see additional electron density in the region where the β-tentacle would be located.

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