Journal:Acta Cryst F:S2053230X22002448

Structure and activity of a thermally stable mutant of Acanthamoeba Actophorin

Stephen Quirk and Raquel Lieberman ^[1]

Molecular Tour
Actophorin is a member of the Actin Depolymerization Factor/Cofilin Family (ADF/C) of proteins that are found in all eukaryotic organisms. The small, monomeric proteins are responsible for severing filamentous actin to help regulate cytokinesis, cell movement, and overall cell architecture. In the amoeba Acanthamoeba castellanii, actophorin facilitates cell movement and is specifically involved with reorganizing the actin network along the leading edge of the amoebastome. This is the appendage that A. castellanii extends from its cell surface to feed. Four actophorin structures can be found in the Protein Data Bank. The original structure, 1AHQ at a resolution of 2.3 Å, a phosphorylated form, 1CNU at 2.3 Å resolution, and a form crystallized in microgravity, 7RTX at 1.7 Å resolution. The fourth structure (the subject of this work) is a mutant form of the protein designed to improve diffraction quality of crystals grown in microgravity environments aboard the International Space Station. The mutant protein contains 19 separate single-point mutations and one C-ter three amino acid deletion. Each mutation individually raises the unfolding transition temperature between 0.5 and 2 oC compared to the wild type protein. When all the mutations are combined into a single protein, it is more stable than wild type actophorin by 22 oC and is still active. The protein crystalized under normal gravity conditions in a new space group; monoclinic P21 versus all the other structures which crystallized in the orthorhombic space group P212121. Crystals of the combined mutant actophorin diffract to 1.7 Å resolution and the structure 7STO, was solved by molecular replacement using 7RTX as a search model. Surprisingly there are two closely associated monomers in the asymmetric unit, but the mutant is not a dimer in solution. The 7STO structure contains four well-coordinated Ni2+ ions, which are critical for crystallization of the mutant, but not for severing activity.

References

1. ↑ Quirk S, Lieberman RL. Structure and activity of a thermally stable mutant of Acanthamoeba actophorin. Acta Crystallogr F Struct Biol Commun. 2022 Apr 1;78(Pt 4):150-160. doi:, 10.1107/S2053230X22002448. Epub 2022 Mar 28. PMID:35400667 doi:http://dx.doi.org/10.1107/S2053230X22002448