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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 identity comparison of CP subunits. The scores of the sequence identity matrices of the CP subunits were rounded and the percentage of sequences plotted against the sequence identity. The inlet contains box plots of the data for each CP subunit. 368 α-subunit and 299 β-subunit CP sequences were derived from CyMoBase [44,45]. For calculating the sequence identities poorly aligned positions and divergent regions of the alignments were removed using Gblocks [46]. Sequence identity matrices (2D-matrix tables containing sequence identities scores for each pair of sequences) were obtained by calculating the ratio of identities to the length of the longer of the two sequences after positions where both sequences contain a gap were removed.
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Figure 3: Sequence identity comparison of CP subunits. The scores of the sequence identity matrices of the CP subunits were rounded and the percentage of sequences plotted against the sequence identity. The inlet contains box plots of the data for each CP subunit. 368 α-subunit and 299 β-subunit CP sequences were derived from CyMoBase [44,45]. For calculating the sequence identities poorly aligned positions and divergent regions of the alignments were removed using Gblocks [46]. Sequence identity matrices (2D-matrix tables containing sequence identities scores for each pair of sequences) were obtained by calculating the ratio of identities to the length of the longer of the two sequences after positions where both sequences contain a gap were removed.

Mentions: Superposition of the Cap32/34 molecule onto its homolog CapZ (PDB code 1IZN) resulted in an r.m.s.d. value of ~1.7 Å over 498 equivalent Cα atoms (the flexible β-subunit C-termini were excluded), which illustrates the highly conserved architecture of the two CP variants (Figure 2A). While the α-subunits of the two homologs superposed with an r.m.s.d. of ~1.7 Å over the Cα atoms (264 residues), the β-subunits match better (r.m.s.d. of ~1.0 Å for 242 residues excluding the β-tentacle), indicative of the latter being structurally more strongly conserved. This is in agreement with findings based on sequence comparisons (Figure 3). In order to quantitatively determine which of the CP subunits is more conserved we calculated sequence identity matrices for all CP subunits in all eukaryotes that have been annotated recently (Hammesfahr and Kollmar, submitted to BMC Evolutionary Biology). Because the data includes sequences from all branches of the eukaryotes each subunit shows a broad distribution. The comparison of the medians of the populations shows that Cap2 (Capβ) is considerably stronger conserved than Cap1 (Capα).


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 identity comparison of CP subunits. The scores of the sequence identity matrices of the CP subunits were rounded and the percentage of sequences plotted against the sequence identity. The inlet contains box plots of the data for each CP subunit. 368 α-subunit and 299 β-subunit CP sequences were derived from CyMoBase [44,45]. For calculating the sequence identities poorly aligned positions and divergent regions of the alignments were removed using Gblocks [46]. Sequence identity matrices (2D-matrix tables containing sequence identities scores for each pair of sequences) were obtained by calculating the ratio of identities to the length of the longer of the two sequences after positions where both sequences contain a gap were removed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Sequence identity comparison of CP subunits. The scores of the sequence identity matrices of the CP subunits were rounded and the percentage of sequences plotted against the sequence identity. The inlet contains box plots of the data for each CP subunit. 368 α-subunit and 299 β-subunit CP sequences were derived from CyMoBase [44,45]. For calculating the sequence identities poorly aligned positions and divergent regions of the alignments were removed using Gblocks [46]. Sequence identity matrices (2D-matrix tables containing sequence identities scores for each pair of sequences) were obtained by calculating the ratio of identities to the length of the longer of the two sequences after positions where both sequences contain a gap were removed.
Mentions: Superposition of the Cap32/34 molecule onto its homolog CapZ (PDB code 1IZN) resulted in an r.m.s.d. value of ~1.7 Å over 498 equivalent Cα atoms (the flexible β-subunit C-termini were excluded), which illustrates the highly conserved architecture of the two CP variants (Figure 2A). While the α-subunits of the two homologs superposed with an r.m.s.d. of ~1.7 Å over the Cα atoms (264 residues), the β-subunits match better (r.m.s.d. of ~1.0 Å for 242 residues excluding the β-tentacle), indicative of the latter being structurally more strongly conserved. This is in agreement with findings based on sequence comparisons (Figure 3). In order to quantitatively determine which of the CP subunits is more conserved we calculated sequence identity matrices for all CP subunits in all eukaryotes that have been annotated recently (Hammesfahr and Kollmar, submitted to BMC Evolutionary Biology). Because the data includes sequences from all branches of the eukaryotes each subunit shows a broad distribution. The comparison of the medians of the populations shows that Cap2 (Capβ) is considerably stronger conserved than Cap1 (Capα).

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