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A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Wong W, Webb AI, Olshina MA, Infusini G, Tan YH, Hanssen E, Catimel B, Suarez C, Condron M, Angrisano F, Nebi T, Kovar DR, Baum J - J. Biol. Chem. (2013)

Bottom Line: Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament.Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing.Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

View Article: PubMed Central - PubMed

Affiliation: From the Divisions of Infection and Immunity and.

ABSTRACT
Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

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The structural basis for PfADF1 and G-actin interactions determined by XL-MS.A, chemical structures of cross-linkers. Sulfo-SDA cross-links lysine to a non-selective amino acid, EDC cross-links lysine to an acidic amino acid (Asp or Glu). B–D, SDS-PAGE gels showing the migration of (B) covalently linked stable complex of monomeric (G)-actin-PfADF1 using EDC and (C) free actin mixed with either GST control or PfADF1 (note between 10 and 25% of actin and PfADF1 are linked by EDC, which is in agreement with their low affinity to form complex). D, interaction between PfADF1 and G-actin analyzed by sulfo-SDA and EDC XL-MS. Three sets of detected sulfo-SDA- and two sets of EDC cross-linked peptides are colored in red, yellow, and blue as highlighted in their primary structures. E, reconstructed XL-MS actin-PfADF1 structural model combining data from cross-linkers sulfo-SDA and EDC. Cross-linked sites by EDC and sulfo-SDA are indicated by white and black lines, respectively. Domains involved in cross-linked peptides are colored (as D). Lysine and acid residues are shown in blue and red spheres, respectively. F, reconstructed G-actin·PfADF1 complex showing basic residue Lys-72 from PfADF1 and acidic residues Glu-99, -100, -361, and -364 from actin. Basic and acidic residues are indicated as blue and red spheres, respectively. Coloring of the G-actin and PfADF1 models and the cross-linked peptides are as described above.
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Figure 2: The structural basis for PfADF1 and G-actin interactions determined by XL-MS.A, chemical structures of cross-linkers. Sulfo-SDA cross-links lysine to a non-selective amino acid, EDC cross-links lysine to an acidic amino acid (Asp or Glu). B–D, SDS-PAGE gels showing the migration of (B) covalently linked stable complex of monomeric (G)-actin-PfADF1 using EDC and (C) free actin mixed with either GST control or PfADF1 (note between 10 and 25% of actin and PfADF1 are linked by EDC, which is in agreement with their low affinity to form complex). D, interaction between PfADF1 and G-actin analyzed by sulfo-SDA and EDC XL-MS. Three sets of detected sulfo-SDA- and two sets of EDC cross-linked peptides are colored in red, yellow, and blue as highlighted in their primary structures. E, reconstructed XL-MS actin-PfADF1 structural model combining data from cross-linkers sulfo-SDA and EDC. Cross-linked sites by EDC and sulfo-SDA are indicated by white and black lines, respectively. Domains involved in cross-linked peptides are colored (as D). Lysine and acid residues are shown in blue and red spheres, respectively. F, reconstructed G-actin·PfADF1 complex showing basic residue Lys-72 from PfADF1 and acidic residues Glu-99, -100, -361, and -364 from actin. Basic and acidic residues are indicated as blue and red spheres, respectively. Coloring of the G-actin and PfADF1 models and the cross-linked peptides are as described above.

Mentions: Attempts to co-crystallize PfADF1 bound to monomeric actin were unsuccessful. To gain a detailed understanding of how PfADF1 interacts with and severs actin filaments, we therefore determined the structural basis of the G-actin-PfADF1 interaction at residue resolution by chemical XL-MS (26–31). This method compliments crystallographic approaches, in that it is able to capture transient interactions and was thus well suited to PfADF1, given its relatively low affinity for actin (Fig. 1E). Recombinant PfADF1 preincubated with monomeric rabbit skeletal muscle actin in the presence of the zero length cross-linking agent 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) yielded a G-actin·PfADF1 complex of ∼56 kDa (Fig. 2, A and B). Incubation of actin with glutathione S-transferase (GST) in the presence of EDC under the same conditions did not yield a protein complex (Fig. 2C). EDC covalently links lysine and acidic residues (Glu or Asp) via a covalent isopeptide bond between the ϵ-amine of lysine and carboxylate group of the acidic residue, allowing fine mapping of interactions mediated by salt bridges. Cross-linked bands were excised, subjected to in-gel tryptic digestion, and analyzed by UPLC and high resolution MS/MS on a Q-Exactive mass spectrometer to detect linked peptides between PfADF1 and actin with the peptide identity searched using a recently developed algorithm to identify cross-linked peptides based on false discovery rate estimation (22). Given the nature of the EDC cross-links, manual validation of the high-resolution spectra permitted mapping of salt bridge interactions between the actin and PfADF1 crystal structures (PDB codes 1J6Z and 3Q2B) with near residue resolution (18, 32). Specifically, SD3 (Lys-328; peptide-(327–335)) of actin was found cross-linked with the coil region preceding α4 (Glu-113; peptide-(101–122)) of PfADF1 (Fig. 2D). In addition, SD3 (Lys-328; peptide-(327–335)) of actin was found cross-linked with the coil region preceding α3 (E81; peptide-(81–86)) of PfADF1 (Fig. 2D). The peptide coverage encompassed most of the PfADF1 C-terminal regions spanning the α3 helix to coil-β6-α4 domains (Fig. 2D). Representative spectra are shown in supplemental data (see supplemental Fig. S1, A and B). Of note, we did not detect any spurious inter-protein cross-links supporting the specificity of this method. Additionally, no peptide or cross-linked identifications were reported when searched with the reversed protein XComb generated database. To further corroborate EDC, we also used sulfo-NHS-diazirine (sulfo-SDA), a short cross-linking agent (3.9 Å), which links lysine residues to non-selective groups (Fig. 2A). Sulfo-SDA cross-linked peptides showed strong spatial overlap to those found with EDC as well as detecting additional cross-linked species. Sulfo-SDA cross-linking revealed PfADF1 residues 81–86, 96–101, and 101–122 bound to subdomain 4 (SD4) (peptide-(211–238)), SD2 (peptide-(51–61)), and SD3 (peptide-(327–335)) of the actin monomer, respectively (Fig. 2D, supplemental Fig. S1, C-E, Table 1, and supplemental Table S1).


A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Wong W, Webb AI, Olshina MA, Infusini G, Tan YH, Hanssen E, Catimel B, Suarez C, Condron M, Angrisano F, Nebi T, Kovar DR, Baum J - J. Biol. Chem. (2013)

The structural basis for PfADF1 and G-actin interactions determined by XL-MS.A, chemical structures of cross-linkers. Sulfo-SDA cross-links lysine to a non-selective amino acid, EDC cross-links lysine to an acidic amino acid (Asp or Glu). B–D, SDS-PAGE gels showing the migration of (B) covalently linked stable complex of monomeric (G)-actin-PfADF1 using EDC and (C) free actin mixed with either GST control or PfADF1 (note between 10 and 25% of actin and PfADF1 are linked by EDC, which is in agreement with their low affinity to form complex). D, interaction between PfADF1 and G-actin analyzed by sulfo-SDA and EDC XL-MS. Three sets of detected sulfo-SDA- and two sets of EDC cross-linked peptides are colored in red, yellow, and blue as highlighted in their primary structures. E, reconstructed XL-MS actin-PfADF1 structural model combining data from cross-linkers sulfo-SDA and EDC. Cross-linked sites by EDC and sulfo-SDA are indicated by white and black lines, respectively. Domains involved in cross-linked peptides are colored (as D). Lysine and acid residues are shown in blue and red spheres, respectively. F, reconstructed G-actin·PfADF1 complex showing basic residue Lys-72 from PfADF1 and acidic residues Glu-99, -100, -361, and -364 from actin. Basic and acidic residues are indicated as blue and red spheres, respectively. Coloring of the G-actin and PfADF1 models and the cross-linked peptides are as described above.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3924271&req=5

Figure 2: The structural basis for PfADF1 and G-actin interactions determined by XL-MS.A, chemical structures of cross-linkers. Sulfo-SDA cross-links lysine to a non-selective amino acid, EDC cross-links lysine to an acidic amino acid (Asp or Glu). B–D, SDS-PAGE gels showing the migration of (B) covalently linked stable complex of monomeric (G)-actin-PfADF1 using EDC and (C) free actin mixed with either GST control or PfADF1 (note between 10 and 25% of actin and PfADF1 are linked by EDC, which is in agreement with their low affinity to form complex). D, interaction between PfADF1 and G-actin analyzed by sulfo-SDA and EDC XL-MS. Three sets of detected sulfo-SDA- and two sets of EDC cross-linked peptides are colored in red, yellow, and blue as highlighted in their primary structures. E, reconstructed XL-MS actin-PfADF1 structural model combining data from cross-linkers sulfo-SDA and EDC. Cross-linked sites by EDC and sulfo-SDA are indicated by white and black lines, respectively. Domains involved in cross-linked peptides are colored (as D). Lysine and acid residues are shown in blue and red spheres, respectively. F, reconstructed G-actin·PfADF1 complex showing basic residue Lys-72 from PfADF1 and acidic residues Glu-99, -100, -361, and -364 from actin. Basic and acidic residues are indicated as blue and red spheres, respectively. Coloring of the G-actin and PfADF1 models and the cross-linked peptides are as described above.
Mentions: Attempts to co-crystallize PfADF1 bound to monomeric actin were unsuccessful. To gain a detailed understanding of how PfADF1 interacts with and severs actin filaments, we therefore determined the structural basis of the G-actin-PfADF1 interaction at residue resolution by chemical XL-MS (26–31). This method compliments crystallographic approaches, in that it is able to capture transient interactions and was thus well suited to PfADF1, given its relatively low affinity for actin (Fig. 1E). Recombinant PfADF1 preincubated with monomeric rabbit skeletal muscle actin in the presence of the zero length cross-linking agent 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) yielded a G-actin·PfADF1 complex of ∼56 kDa (Fig. 2, A and B). Incubation of actin with glutathione S-transferase (GST) in the presence of EDC under the same conditions did not yield a protein complex (Fig. 2C). EDC covalently links lysine and acidic residues (Glu or Asp) via a covalent isopeptide bond between the ϵ-amine of lysine and carboxylate group of the acidic residue, allowing fine mapping of interactions mediated by salt bridges. Cross-linked bands were excised, subjected to in-gel tryptic digestion, and analyzed by UPLC and high resolution MS/MS on a Q-Exactive mass spectrometer to detect linked peptides between PfADF1 and actin with the peptide identity searched using a recently developed algorithm to identify cross-linked peptides based on false discovery rate estimation (22). Given the nature of the EDC cross-links, manual validation of the high-resolution spectra permitted mapping of salt bridge interactions between the actin and PfADF1 crystal structures (PDB codes 1J6Z and 3Q2B) with near residue resolution (18, 32). Specifically, SD3 (Lys-328; peptide-(327–335)) of actin was found cross-linked with the coil region preceding α4 (Glu-113; peptide-(101–122)) of PfADF1 (Fig. 2D). In addition, SD3 (Lys-328; peptide-(327–335)) of actin was found cross-linked with the coil region preceding α3 (E81; peptide-(81–86)) of PfADF1 (Fig. 2D). The peptide coverage encompassed most of the PfADF1 C-terminal regions spanning the α3 helix to coil-β6-α4 domains (Fig. 2D). Representative spectra are shown in supplemental data (see supplemental Fig. S1, A and B). Of note, we did not detect any spurious inter-protein cross-links supporting the specificity of this method. Additionally, no peptide or cross-linked identifications were reported when searched with the reversed protein XComb generated database. To further corroborate EDC, we also used sulfo-NHS-diazirine (sulfo-SDA), a short cross-linking agent (3.9 Å), which links lysine residues to non-selective groups (Fig. 2A). Sulfo-SDA cross-linked peptides showed strong spatial overlap to those found with EDC as well as detecting additional cross-linked species. Sulfo-SDA cross-linking revealed PfADF1 residues 81–86, 96–101, and 101–122 bound to subdomain 4 (SD4) (peptide-(211–238)), SD2 (peptide-(51–61)), and SD3 (peptide-(327–335)) of the actin monomer, respectively (Fig. 2D, supplemental Fig. S1, C-E, Table 1, and supplemental Table S1).

Bottom Line: Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament.Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing.Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

View Article: PubMed Central - PubMed

Affiliation: From the Divisions of Infection and Immunity and.

ABSTRACT
Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

Show MeSH
Related in: MedlinePlus