7SOG

Thermostable actophorin

Summary for 7SOG

• Entry DOI: 10.2210/pdb7sog/pdb
• Descriptor: Actophorin, NICKEL (II) ION (3 entities in total)
• Functional Keywords: motor protein, mutagenesis
• Biological source: Acanthamoeba castellanii (Amoeba)
• Total number of polymer chains: 2
• Total formula weight: 30601.07

Authors

Lieberman, R.L.,Quirk, S. (deposition date: 2021-10-30, release date: 2022-04-20, Last modification date: 2023-10-18)

Primary citation

Quirk, S.,Lieberman, R.L.
Structure and activity of a thermally stable mutant of Acanthamoeba actophorin.
Acta Crystallogr.,Sect.F, 78:150-160, 2022

PubMed Abstract: Actophorin, which was recently tested for crystallization under microgravity on the International Space Station, was subjected to mutagenesis to identify a construct with improved biophysical properties that were expected to improve the extent of diffraction. First, 20 mutations, including one C-terminal deletion of three residues, were introduced individually into actophorin, resulting in modest increases in thermal stability of between +0.5°C and +2.2°C. All but two of the stabilizing mutants increased both the rates of severing F-actin filaments and of spontaneous polymerization of pyrenyl G-actin in vitro. When the individual mutations were combined into a single actophorin variant, Acto-2, the overall thermal stability was 22°C higher than that of wild-type actophorin. When an inactivating S2P mutation in Acto-2 was restored, Acto-2/P2S was more stable by 20°C but was notably more active than the wild-type protein. The inactivating S2P mutation reaffirms the importance that Ser2 plays in the F-actin-severing reaction. The crystal structure of Acto-2 was solved to 1.7 Å resolution in a monoclinic space group, a first for actophorin. Surprisingly, despite the increase in thermal stability, the extended β-turn region, which is intimately involved in interactions with F-actin, is disordered in one copy of Acto-2 in the asymmetric unit. These observations emphasize the complex interplay among protein thermal stability, function and dynamics.

PubMed: 35400667
DOI: 10.1107/S2053230X22002448

Experimental method

X-RAY DIFFRACTION (1.74 Å)