Limits...
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.

Show MeSH

Related in: MedlinePlus

Sequence conservation within the actin-binding region of the α-subunits. The sequence logos are based on 368 α-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the α-subunits. Here, only the C-termini of the α-subunits are shown because most of the residues implicated in actin binding map to this region (For the representation of the entire α-subunits see Additional file 1). For better orientation, the sequences of five representative α-subunits are shown: the three isoforms of chicken Cap1 for comparison because all previous crystal structures have been obtained from chicken Cap1α, the yeast Cap1 as one of the targets of mutagenesis experiments, and Dictyostelium Cap34 whose structure is presented here. Secondary structural elements as determined from the chicken CapZ crystal structure are drawn as yellow arrows (β-strands) and as red boxes (α-helices). Residues important for inter-heterodimer binding, V-1 binding, PIP2-binding, and actin-binding are highlighted by orange, green, red, and purple stars, respectively. Numbering below the logos refers to positions in the multiple sequence alignment (The full-length multiple sequence alignment of the α-subunits is available as Additional File 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472329&req=5

Figure 4: Sequence conservation within the actin-binding region of the α-subunits. The sequence logos are based on 368 α-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the α-subunits. Here, only the C-termini of the α-subunits are shown because most of the residues implicated in actin binding map to this region (For the representation of the entire α-subunits see Additional file 1). For better orientation, the sequences of five representative α-subunits are shown: the three isoforms of chicken Cap1 for comparison because all previous crystal structures have been obtained from chicken Cap1α, the yeast Cap1 as one of the targets of mutagenesis experiments, and Dictyostelium Cap34 whose structure is presented here. Secondary structural elements as determined from the chicken CapZ crystal structure are drawn as yellow arrows (β-strands) and as red boxes (α-helices). Residues important for inter-heterodimer binding, V-1 binding, PIP2-binding, and actin-binding are highlighted by orange, green, red, and purple stars, respectively. Numbering below the logos refers to positions in the multiple sequence alignment (The full-length multiple sequence alignment of the α-subunits is available as Additional File 2).

Mentions: Like in CapZ’s α-subunit the C-terminus of Cap34 includes a short amphipathic α-helix (also called α-tentacle), which is tightly connected by hydrophobic contacts to the body of the β-subunit through a strictly conserved tryptophan residue (Trp-267 in Cap34 from Dictyostelium discoideum, Trp-271 in chicken CapZ; Figure 4). The α-tentacle is bound to the β-subunit of CP in all crystal and NMR structures. Especially the NMR analyses show that the flexibility of the α-subunit’s C-terminus is limited to the last 12 residues (L275 – A286 in human Cap1α), which are C-terminal to the strictly conserved tryptophan residue and the 1-turn helix [48,49]. In addition, the C-terminal truncation mutants mouse Cap1α ΔC13 [50] and yeast Cap1α ΔC10 [51] showed only a weak effect on actin binding as did many single residue mutations in the C-terminus of yeast Cap1 [51]. In contrast, longer C-terminal truncations of 28 (mouse Cap1α ΔC28; [50,52]) and 30 residues (yeast Cap1 ΔC30; [51]) abolished actin-binding. In view of the tight and conserved interaction of the antiparallel helices with the central β-sheet the effects of the longer C-terminal truncations could also be due to the disturbance of the structural stability of this region. Thus the α-subunit’s interaction with actin is either solely mediated by the basic patch, in which case the α-tentacle would not move but retain the integrity and stability of the CP dimer, or the α-tentacle moves out of its position to bind actin thus opening a hydrophobic patch on the CP surface. These possibilities can only be tested by mutations that do not disturb the stability of this region. Based on the NMR experiments, the results from the short C-terminal truncations, and the many single residues mutations in the α-tentacle it seems most likely that the α-tentacle is not moving upon actin-binding. The only flexible region consists of the C-terminal 12 residues, which, however, are not strongly conserved and only show a slight effect on actin-binding.


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 α-subunits. The sequence logos are based on 368 α-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the α-subunits. Here, only the C-termini of the α-subunits are shown because most of the residues implicated in actin binding map to this region (For the representation of the entire α-subunits see Additional file 1). For better orientation, the sequences of five representative α-subunits are shown: the three isoforms of chicken Cap1 for comparison because all previous crystal structures have been obtained from chicken Cap1α, the yeast Cap1 as one of the targets of mutagenesis experiments, and Dictyostelium Cap34 whose structure is presented here. Secondary structural elements as determined from the chicken CapZ crystal structure are drawn as yellow arrows (β-strands) and as red boxes (α-helices). Residues important for inter-heterodimer binding, V-1 binding, PIP2-binding, and actin-binding are highlighted by orange, green, red, and purple stars, respectively. Numbering below the logos refers to positions in the multiple sequence alignment (The full-length multiple sequence alignment of the α-subunits is available as Additional File 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sequence conservation within the actin-binding region of the α-subunits. The sequence logos are based on 368 α-subunit sequences and illustrate the sequence conservation within the multiple sequence alignment of the α-subunits. Here, only the C-termini of the α-subunits are shown because most of the residues implicated in actin binding map to this region (For the representation of the entire α-subunits see Additional file 1). For better orientation, the sequences of five representative α-subunits are shown: the three isoforms of chicken Cap1 for comparison because all previous crystal structures have been obtained from chicken Cap1α, the yeast Cap1 as one of the targets of mutagenesis experiments, and Dictyostelium Cap34 whose structure is presented here. Secondary structural elements as determined from the chicken CapZ crystal structure are drawn as yellow arrows (β-strands) and as red boxes (α-helices). Residues important for inter-heterodimer binding, V-1 binding, PIP2-binding, and actin-binding are highlighted by orange, green, red, and purple stars, respectively. Numbering below the logos refers to positions in the multiple sequence alignment (The full-length multiple sequence alignment of the α-subunits is available as Additional File 2).
Mentions: Like in CapZ’s α-subunit the C-terminus of Cap34 includes a short amphipathic α-helix (also called α-tentacle), which is tightly connected by hydrophobic contacts to the body of the β-subunit through a strictly conserved tryptophan residue (Trp-267 in Cap34 from Dictyostelium discoideum, Trp-271 in chicken CapZ; Figure 4). The α-tentacle is bound to the β-subunit of CP in all crystal and NMR structures. Especially the NMR analyses show that the flexibility of the α-subunit’s C-terminus is limited to the last 12 residues (L275 – A286 in human Cap1α), which are C-terminal to the strictly conserved tryptophan residue and the 1-turn helix [48,49]. In addition, the C-terminal truncation mutants mouse Cap1α ΔC13 [50] and yeast Cap1α ΔC10 [51] showed only a weak effect on actin binding as did many single residue mutations in the C-terminus of yeast Cap1 [51]. In contrast, longer C-terminal truncations of 28 (mouse Cap1α ΔC28; [50,52]) and 30 residues (yeast Cap1 ΔC30; [51]) abolished actin-binding. In view of the tight and conserved interaction of the antiparallel helices with the central β-sheet the effects of the longer C-terminal truncations could also be due to the disturbance of the structural stability of this region. Thus the α-subunit’s interaction with actin is either solely mediated by the basic patch, in which case the α-tentacle would not move but retain the integrity and stability of the CP dimer, or the α-tentacle moves out of its position to bind actin thus opening a hydrophobic patch on the CP surface. These possibilities can only be tested by mutations that do not disturb the stability of this region. Based on the NMR experiments, the results from the short C-terminal truncations, and the many single residues mutations in the α-tentacle it seems most likely that the α-tentacle is not moving upon actin-binding. The only flexible region consists of the C-terminal 12 residues, which, however, are not strongly conserved and only show a slight effect on actin-binding.

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