Solution structure and dynamics of ADF from Toxoplasma gondii

Abstract

Toxoplasma gondii (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from 15N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin with the G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the typical F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B fromCaenorhabditis elegans

“…Residues of α3-β6 loop (L133, T135) of UNC-60A and (G122, L123, L126) of UNC-60B show conformational flexibility on the millisecond to microsecond time scale in both proteins, and similar dynamic behaviour for this region was also observed for TgADF [27]. In the F-loop mutant K91A of UNC-60B, in addition to residues of F-site, some residues of α3-β6 loop also show CSPs.…”

“…Amongst all the known ADF/cofilin structures, structures of PfADF1 and TgADF display the shortest F-loop that does not protrude out of the structure [27, 46]. In both these proteins, there is a single basic residue (K72 in PfADF1; K68 in TgADF), which is essential for their weak severing activity.…”

“…In UNC-60B fourteen residues of F-loop show motion on nanosecond-picosecond timescale. In weak severing proteins like UNC-60A and LdCof, seven and nine salt bridges are formed by the residues of F-loop with the residues of other regions, respectively [27]. In strong severing proteins like UNC-60B, yeast cofilin, Acanthamoeba actophorin, human cofilin and chick cofilin only one to three salt bridges are formed by the residues of F-loop with the residues of other regions [2, 12, 35, 36, 37] [Table 2].…”

“…The ITC experiments were performed by adding aliquots of ligands to rabbit muscle actin. The rabbit muscle actin for the experiments and samples for ITC (Stock solution of UNC-60A, UNC-60B and rabbit muscle ADP-G-actin) were prepared as described earlier [26, 27]. The sample cell was filled with 1.459 mL of 0.01 mM of rabbit muscle actin (titrand) and titrated against UNC-60A which was filled in the syringe of 290 μL at a concentration of 0.100 mM.…”

The other side of the coin: Functional and structural versatility of ADF/cofilins

“…(human platelet) stimulation (human platelet) (Singh et al, 2011) T. gondii TgADF YES/? (RbskACT, TgACT) stimulation at low concentrations (< 1:1) (RbskACT, TgACT) (Mehta and Sibley, 2010;Yadav et al, 2011) inhibition at high concentration (> 1:1) (RbskACT, TgACT)…”