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.
Highlights
Plasmodium falciparum actin depolymerizing factor 1 (PfADF1) severs actin polymers without stable filament-binding, challenging current models for severing
Despite the inability of PfADF1 to alter the pitch of the actin polymer, measurement of filament lengths by transmission electron microscopy (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 total internal reflection fluorescence (TIRF) microscopy 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 Actin depolymerizing factor (ADF)/ cofilin from the apicomplexan parasite Toxoplasma gondii (15)
Summary
Plasmodium falciparum actin depolymerizing factor 1 (PfADF1) severs actin polymers without stable filament-binding, challenging current models for severing. 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. Single-celled protozoan pathogens that include the etiological agents of malaria and toxoplasmosis, ADF/cofilin proteins display unusual properties in their regulation and interaction with the actin filament (14 –18). They are able to sever filaments yet possess low binding affinity for F-actin (15, 18). Propose that the novel mechanism used by malaria parasite PfADF1 is potentially employed by diverse ADF/cofilins to mediate rapid F-actin turnover
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