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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 HsCOF1-F-actin interaction determined by EDC XL-MS.A, interaction between HsCOF1 and F-actin at the decoration site determined by EDC XL-MS. Four sets of detected EDC-cross-linked peptides corresponding to actin-binding site 1 are colored in blue, purple, red, and orange as highlighted in their primary structures. B, four sets of EDC cross-linked F-actin-HsCOF1 peptides were mapped onto the cryo-EM structure of the HsCOF2 decorated filament. Colors are as described in A. Cross-linked sites are indicated by white lines.
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Figure 4: The structural basis for HsCOF1-F-actin interaction determined by EDC XL-MS.A, interaction between HsCOF1 and F-actin at the decoration site determined by EDC XL-MS. Four sets of detected EDC-cross-linked peptides corresponding to actin-binding site 1 are colored in blue, purple, red, and orange as highlighted in their primary structures. B, four sets of EDC cross-linked F-actin-HsCOF1 peptides were mapped onto the cryo-EM structure of the HsCOF2 decorated filament. Colors are as described in A. Cross-linked sites are indicated by white lines.

Mentions: We then attempted to use XL-MS to characterize the interaction between F-actin and a conventional ADF/cofilin, HsCOF1. EDC XL-MS of HsCOF1 bound to F-actin revealed four cross-linked peptides corresponding to the decorative interaction interface as described in detail previously (9) and entirely consistence with the sites of interactions observed in the cryo-EM structure of the F-actin·HsCOF2 complex (9). Specifically, SD3 of actin protomer n+2 (Lys-328 from peptide-(327–335) and Lys-291 from peptide-(291–312)) were found interacting with Glu-142 (peptide-(133–144)) and Glu-151 (peptide-(147–152)) of HsCOF1, respectively (Fig. 4, A and B, supplemental Fig. S3, A and B, Table 3, and supplemental Table S1). For actin protomer n, HsCOF1 was found to interact with Asp-25 of SD1 (peptide-(19–28)) via Lys-95 (peptide-(93–112)), whereas Glu-51 of SD2 (peptide-(51–62)) was found to interact with Lys-125 (peptide-(122–127)) (Fig. 4, A and B, supplemental Fig. S3, A-E, Table 3, 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 HsCOF1-F-actin interaction determined by EDC XL-MS.A, interaction between HsCOF1 and F-actin at the decoration site determined by EDC XL-MS. Four sets of detected EDC-cross-linked peptides corresponding to actin-binding site 1 are colored in blue, purple, red, and orange as highlighted in their primary structures. B, four sets of EDC cross-linked F-actin-HsCOF1 peptides were mapped onto the cryo-EM structure of the HsCOF2 decorated filament. Colors are as described in A. Cross-linked sites are indicated by white lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The structural basis for HsCOF1-F-actin interaction determined by EDC XL-MS.A, interaction between HsCOF1 and F-actin at the decoration site determined by EDC XL-MS. Four sets of detected EDC-cross-linked peptides corresponding to actin-binding site 1 are colored in blue, purple, red, and orange as highlighted in their primary structures. B, four sets of EDC cross-linked F-actin-HsCOF1 peptides were mapped onto the cryo-EM structure of the HsCOF2 decorated filament. Colors are as described in A. Cross-linked sites are indicated by white lines.
Mentions: We then attempted to use XL-MS to characterize the interaction between F-actin and a conventional ADF/cofilin, HsCOF1. EDC XL-MS of HsCOF1 bound to F-actin revealed four cross-linked peptides corresponding to the decorative interaction interface as described in detail previously (9) and entirely consistence with the sites of interactions observed in the cryo-EM structure of the F-actin·HsCOF2 complex (9). Specifically, SD3 of actin protomer n+2 (Lys-328 from peptide-(327–335) and Lys-291 from peptide-(291–312)) were found interacting with Glu-142 (peptide-(133–144)) and Glu-151 (peptide-(147–152)) of HsCOF1, respectively (Fig. 4, A and B, supplemental Fig. S3, A and B, Table 3, and supplemental Table S1). For actin protomer n, HsCOF1 was found to interact with Asp-25 of SD1 (peptide-(19–28)) via Lys-95 (peptide-(93–112)), whereas Glu-51 of SD2 (peptide-(51–62)) was found to interact with Lys-125 (peptide-(122–127)) (Fig. 4, A and B, supplemental Fig. S3, A-E, Table 3, 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