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Structural and functional studies on Plasmodium formins

Final Report Summary - FORMIN (Structural and functional studies on Plasmodium formins)

Executive Summary:

Project Context and Objectives:

The overall aim of this project was to gather structural and functional information on the profilin-formin-mediated actin polymerization in the malaria parasite. Specifically, the aims were:

- to express, purify, and crystallize the FH1-FH2 domains of the two Plasmodium formin isoforms alone and in binary and ternary complexes with actin and profilin
- to characterize the binding of both formins to actin and profilin
- to characterize the biochemical activities of the formins on actin and their possible co-operativity with profilin

Several constructs of Plasmodium formins 1 and 2 have been cloned from genomic DNA into bacterial expression vectors. Soluble expression conditions have been established for several constructs of both formins, including the FH1-FH2 domains together and separately. These proteins have been purified in large scale, crystallization screening has been started, and the differences of the different domains in actin nucleation in the presence and absence of profilin have been characterized using fluorescence spectroscopic methods. In addition, small-angle scattering of X-rays (SAXS) and neutrons (SANS) has been used for low-resolution structure determination of the domains in solution, and it was shown that compact dimerization only occurs in the presence of the lasso segment and is required for nucleation activity. The first results of this project were published in early 2012 (Ignatev et al. 2012, PLoS ONE).

Both Plasmodium formins have been shown by electron microscopy to bundle actin filaments. Interestingly, the formin 1 induced bundles appear thicker and tighter than the formin 2 induced ones. Formin 2 was was also shown to form a complex with Plasmodium profilin, and the structural characterization of this complex is underway. No interaction of the rudimentary FH1 domain of formin 1 could be shown with Plasmodium profilin. Formin 1 has also been shown to nucleate filaments of recombinantly expressed Plasmodium actins. However, formin-induced bundles have only been observed of the minor isoform Plasmodium actin II and not of actin I. This may be due to the different filament structures of the two Plasmodium actin isoforms.

Future work will be focused more on crystallization of the formins and complex formation and characterization of formins, actin, and profilin as well as cryo-EM on formins bound to filamentous actin.