Cryo-EM structure of ASK1 suggests a role of inter-domain interactions and TRX1 in its regulation

K. Honzejkova¹, D. Kosek², V. Obsilova², T. Obsil^1,2

¹ Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic

² Department of Structural Biology of Signalling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic

karolina.honzejkova@natur.cuni.cz

ASK1 (apoptosis signal-regulating kinase 1) is a member of MAP3K protein family, which directs cells towards inflammation or apoptosis through p38 or JNK signalling pathway [1,2]. Given its involvement in several serious diseases, ASK1 has been considered a promising target for therapeutic intervention [3]. Unfortunately, lack of relevant structural data and insufficient understanding of the intricate regulation of the kinase both hamper the search for effective drugs.

In this study, we employed several methods to expand our understanding of ASK1 regulation and the role of thioredoxin 1 in its inhibition. Firstly, AUC showed that C-terminally truncated ASK1 tends to dimerize in solution and thioredoxin 1 appears to further promote this tendency. HDX-MS then allowed us to map the contacts between individual domains within a protomer as well as the dimerization interface. Also, it allowed us to identify the regions affected by thioredoxin 1 binding. Finally, single-particle cryo-EM analysis enabled us to determine the first structure of the C-terminally truncated ASK1 in its dimeric form.

Collectively, our results provide the first structural insight into dimeric ASK1 and suggest how binding partners might affect its activity. Presented findings may serve as a basis for the design of ASK1-specific inhibitors and represent a starting point for further experiments aiming at even fuller understanding of ASK1 regulation and functioning.

1. H. Ichijo, E. Nishida, K. Irie, P. Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumuto, K. Miyazono, Y. Gotoh, Science, 275, (1997), 90-94.

2. K. Tobiume, A. Matsazuwa, T. Takahashi, H. Nishitoh, K. Morita, K. Takeda, O. Minowa, K. Miyazono, T. Noda, H. Ichijo, EMBO Rep, 2, (2001), 222-228.

3. V. Obsilova, K. Honzejkova, T. Obsilova, Int J Mo. Sci, 22, (2021), 13395.

This study was supported by the Czech Science Foundation (Project 19-00121S). We acknowledge CMS-Biocev ("Biophysical techniques, Crystallization, Diffraction, Structural mass spectrometry”) of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2018127) and CZ.02.1.01/0.0/0.0/18_046/0015974 and Cryo-electron microscopy and tomography core facility CEITEC MU of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042) and European Regional Development Fund-Project „UP CIISB“ (No. CZ.02.1.01/0.0/0.0/18_046/0015974).