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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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PfADF1 interaction with monomeric and filamentous actin.A, negative stain and TEM of F-actin with and without ADF/cofilin proteins. Measurements shown are in nm, with examples of pitch highlighted. Scale bars, 50 nm. B, measurement of filament pitch by TEM (mean ± S.E.). C, measurement of filament length by TEM (mean ± S.E.). D, sedimentation analysis of PfADF1 and PfADF1.K72A in preventing the formation of long filaments. G-actin (2 μm) was incubated with increasing concentrations of ADF proteins for 1 h before ultracentrifugation to separate short actin species (Supernatant) and polymerized F-actin (pellet). Equal amounts of supernatant and pellet fractions were assessed by SDS-PAGE and the percentage of actin in each fraction was analyzed by densitometry (n = 3, mean ± S.E.). E, measurement of binding affinities between G-actin and PfADF1 or PfADF1.K72A derivatives using surface plasma resonance. The experimental data were analyzed using a 1:1 interaction model using the rate equation described in BIAevaluation software 4.1. Data were fitted using a global analysis module that constrains selected parameters (ka and kd) to a single solution for the all set of binding curves, improving the robustness of the fitting procedure. This set of rate constants was used for calculation of the equilibrium dissociation constant (KD = kd/ka). F, model of published crystal structure of the Twinfilin·ADFH·actin complex (PDB code 3DAW). Serine 252 of Twinfilin-ADFH is shown as a green sphere, which is the equivalent residue to Lys-72 of PfADF1.
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Figure 1: PfADF1 interaction with monomeric and filamentous actin.A, negative stain and TEM of F-actin with and without ADF/cofilin proteins. Measurements shown are in nm, with examples of pitch highlighted. Scale bars, 50 nm. B, measurement of filament pitch by TEM (mean ± S.E.). C, measurement of filament length by TEM (mean ± S.E.). D, sedimentation analysis of PfADF1 and PfADF1.K72A in preventing the formation of long filaments. G-actin (2 μm) was incubated with increasing concentrations of ADF proteins for 1 h before ultracentrifugation to separate short actin species (Supernatant) and polymerized F-actin (pellet). Equal amounts of supernatant and pellet fractions were assessed by SDS-PAGE and the percentage of actin in each fraction was analyzed by densitometry (n = 3, mean ± S.E.). E, measurement of binding affinities between G-actin and PfADF1 or PfADF1.K72A derivatives using surface plasma resonance. The experimental data were analyzed using a 1:1 interaction model using the rate equation described in BIAevaluation software 4.1. Data were fitted using a global analysis module that constrains selected parameters (ka and kd) to a single solution for the all set of binding curves, improving the robustness of the fitting procedure. This set of rate constants was used for calculation of the equilibrium dissociation constant (KD = kd/ka). F, model of published crystal structure of the Twinfilin·ADFH·actin complex (PDB code 3DAW). Serine 252 of Twinfilin-ADFH is shown as a green sphere, which is the equivalent residue to Lys-72 of PfADF1.

Mentions: The malaria parasite P. falciparum ADF/cofilin (PfADF1) has low binding affinity for actin filaments (15, 18). Given PfADF1 lacks the critical motif (the filament binding loop and α4 helix comprising the F-site) thought to be required for canonical decoration, we hypothesized PfADF1 may not be able to reduce the pitch of actin filaments. To test this hypothesis, we directly visualized polymerized rabbit skeletal muscle actin by transmission electron microscopy (TEM) in the presence or absence of recombinant PfADF1 or the canonical ADF/cofilin HsCOF1, which retains an intact F-site (9). In the absence of ADF/cofilin binding, filaments demonstrated a typical ∼39.8 nm pitch (n = 23, Fig. 1, A and B) (8). Following incubation with 10 μm HsCOF1, filament pitch was reduced to a 26.3 nm mean, confirming known dimensions of the twisted HsCOF1-decorated filament (Fig. 1, A and B) (8). Similar concentrations of PfADF1, known to be active for severing (18), demonstrated no significant reduction in pitch (mean 39.4, n = 20, Fig. 1, A and B). Apicomplexan ADF/cofilin proteins thus appear unable to alter the pitch of F-actin. Despite the inability of PfADF1 to alter the pitch of the actin polymer, measurement of filament lengths by TEM showed that both HsCOF1 and PfADF1 efficiently reduced F-actin length, with a 3-fold reduction in polymer length for both proteins compared with untreated filaments (Fig. 1C). The ability of PfADF1 to sever was confirmed by TIRF microscopy (supplemental Movies S1 and S2) and the reduction in lengths seen are entirely consistent with previous reports of comparable severing rates, as measured by TIRF, between PfADF1 and HsCOF1 (18) along with a related ADF/cofilin from the apicomplexan parasite Toxoplasma gondii (15). This therefore demonstrates that actin filament severing does not require stable decoration of F-actin by PfADF1 and suggests that an alternative mechanism may exist that underpins PfADF1-mediated severing.


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)

PfADF1 interaction with monomeric and filamentous actin.A, negative stain and TEM of F-actin with and without ADF/cofilin proteins. Measurements shown are in nm, with examples of pitch highlighted. Scale bars, 50 nm. B, measurement of filament pitch by TEM (mean ± S.E.). C, measurement of filament length by TEM (mean ± S.E.). D, sedimentation analysis of PfADF1 and PfADF1.K72A in preventing the formation of long filaments. G-actin (2 μm) was incubated with increasing concentrations of ADF proteins for 1 h before ultracentrifugation to separate short actin species (Supernatant) and polymerized F-actin (pellet). Equal amounts of supernatant and pellet fractions were assessed by SDS-PAGE and the percentage of actin in each fraction was analyzed by densitometry (n = 3, mean ± S.E.). E, measurement of binding affinities between G-actin and PfADF1 or PfADF1.K72A derivatives using surface plasma resonance. The experimental data were analyzed using a 1:1 interaction model using the rate equation described in BIAevaluation software 4.1. Data were fitted using a global analysis module that constrains selected parameters (ka and kd) to a single solution for the all set of binding curves, improving the robustness of the fitting procedure. This set of rate constants was used for calculation of the equilibrium dissociation constant (KD = kd/ka). F, model of published crystal structure of the Twinfilin·ADFH·actin complex (PDB code 3DAW). Serine 252 of Twinfilin-ADFH is shown as a green sphere, which is the equivalent residue to Lys-72 of PfADF1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: PfADF1 interaction with monomeric and filamentous actin.A, negative stain and TEM of F-actin with and without ADF/cofilin proteins. Measurements shown are in nm, with examples of pitch highlighted. Scale bars, 50 nm. B, measurement of filament pitch by TEM (mean ± S.E.). C, measurement of filament length by TEM (mean ± S.E.). D, sedimentation analysis of PfADF1 and PfADF1.K72A in preventing the formation of long filaments. G-actin (2 μm) was incubated with increasing concentrations of ADF proteins for 1 h before ultracentrifugation to separate short actin species (Supernatant) and polymerized F-actin (pellet). Equal amounts of supernatant and pellet fractions were assessed by SDS-PAGE and the percentage of actin in each fraction was analyzed by densitometry (n = 3, mean ± S.E.). E, measurement of binding affinities between G-actin and PfADF1 or PfADF1.K72A derivatives using surface plasma resonance. The experimental data were analyzed using a 1:1 interaction model using the rate equation described in BIAevaluation software 4.1. Data were fitted using a global analysis module that constrains selected parameters (ka and kd) to a single solution for the all set of binding curves, improving the robustness of the fitting procedure. This set of rate constants was used for calculation of the equilibrium dissociation constant (KD = kd/ka). F, model of published crystal structure of the Twinfilin·ADFH·actin complex (PDB code 3DAW). Serine 252 of Twinfilin-ADFH is shown as a green sphere, which is the equivalent residue to Lys-72 of PfADF1.
Mentions: The malaria parasite P. falciparum ADF/cofilin (PfADF1) has low binding affinity for actin filaments (15, 18). Given PfADF1 lacks the critical motif (the filament binding loop and α4 helix comprising the F-site) thought to be required for canonical decoration, we hypothesized PfADF1 may not be able to reduce the pitch of actin filaments. To test this hypothesis, we directly visualized polymerized rabbit skeletal muscle actin by transmission electron microscopy (TEM) in the presence or absence of recombinant PfADF1 or the canonical ADF/cofilin HsCOF1, which retains an intact F-site (9). In the absence of ADF/cofilin binding, filaments demonstrated a typical ∼39.8 nm pitch (n = 23, Fig. 1, A and B) (8). Following incubation with 10 μm HsCOF1, filament pitch was reduced to a 26.3 nm mean, confirming known dimensions of the twisted HsCOF1-decorated filament (Fig. 1, A and B) (8). Similar concentrations of PfADF1, known to be active for severing (18), demonstrated no significant reduction in pitch (mean 39.4, n = 20, Fig. 1, A and B). Apicomplexan ADF/cofilin proteins thus appear unable to alter the pitch of F-actin. Despite the inability of PfADF1 to alter the pitch of the actin polymer, measurement of filament lengths by TEM showed that both HsCOF1 and PfADF1 efficiently reduced F-actin length, with a 3-fold reduction in polymer length for both proteins compared with untreated filaments (Fig. 1C). The ability of PfADF1 to sever was confirmed by TIRF microscopy (supplemental Movies S1 and S2) and the reduction in lengths seen are entirely consistent with previous reports of comparable severing rates, as measured by TIRF, between PfADF1 and HsCOF1 (18) along with a related ADF/cofilin from the apicomplexan parasite Toxoplasma gondii (15). This therefore demonstrates that actin filament severing does not require stable decoration of F-actin by PfADF1 and suggests that an alternative mechanism may exist that underpins PfADF1-mediated severing.

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