The cooperative folding of annexin A2 relies on a transient nonnative intermediate.

Hanne Hollås, Juan Ramirez, Yves Nominé, Camille Kostmann, Angelo Toto, Stefano Gianni, Gilles Travé, Anni Vedeler

Biophysical journal 2022 Dec 06

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Annexins (Anxs) are a family of highly homologous proteins that bind and aggregate lipid vesicles in the presence of calcium. All members of the family contain a variable N-terminus determining specific functions, followed by a conserved core region responsible for the general calcium-dependent lipid-binding property. The core structure consists of four homologous domains (DI-DIV), each consisting of a right-handed super-helix of five α-helices. We present data from a combination of site-directed mutagenesis, NMR, and circular dichroism showing that the G25-D34 region of the N-terminus as well as the contacts between residues D38A, R63A, and Q67A of AnxA2-DI are crucial for the autonomous folding and stability of DI of AnxA2. However, we also show that the folding of the full-length protein is very robust in that mutations and truncations that disrupted the folding of AnxA2-DI did not abolish the folding of full-length AnxA2, only lowering its thermal stability. This robustness of the folding of full-length AnxA2 is likely to be mediated by the existence of at least one transient nonnative intermediate as suggested by our kinetic data using stopped-flow fluorescence experiments. We also show that hydrophobic amino acids in AnxA2-DI involved in interfacial contacts with AnxA2-DIV are important for the cooperative folding and stability of the full-length protein. Mutating all of the V57E-V98R-G101Y residues in AnxA2-DI did not affect the folding of the domain, only its stability, but prevented the cooperative folding of the full-length protein. Our collective results favor a highly cooperative and robust folding process mediated by alternative intermediate steps. Since AnxA2 is a multifunctional protein involved in several steps of the progression of cell transformation, these data on structure and folding pathways are therefore crucial to designing anticancer drugs targeting AnxA2. Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Citation

Hanne Hollås, Juan Ramirez, Yves Nominé, Camille Kostmann, Angelo Toto, Stefano Gianni, Gilles Travé, Anni Vedeler. The cooperative folding of annexin A2 relies on a transient nonnative intermediate. Biophysical journal. 2022 Dec 06;121(23):4492-4504