Oral Presentation 23rd International Society of Magnetic Resonance Conference 2023

NMR characterization of the amyloid forming protein RIPK3 reveals a cryptic region for self-recognition (#155)

Paula Polonio 1 , Gustavo Alfredo Titaux-Delgado 1 , Nikhil R. Varghese 2 , Chi L. L. Pham 2 , Karyn Wilde 3 , Margie Sunde 2 , Miguel Mompeán 1
  1. Rocasolano Institute of Physical Chemistry, Spanish National Research Council, Madrid, Spain
  2. School of Medical Sciences, Sydney Nano and Sydney Infectious Diseases, Sydney, NSW, Australia
  3. National Deuteration Facility Australian Nuclear Science and Technology Organization (ANSTO), Sydney, NSW, Australia

RIPK3 is a receptor-interacting protein kinase that harbors a RIP homotypic interaction motif (RHIM). RHIMs are involved in inflammatory and cell death signaling, promoting the assembly of functional amyloids (1), but also leading to pathological amyloid formation under certain circumstances (2). Although the amyloid-bound form of RIPK3 has been already characterized by cryo-EM and solid-state NMR (3,4), the dynamics and the structural information of RHIM-containing proteins like RIPK3 prior assembly are difficult to obtain due to their natural tendency to aggregate, and therefore remain unknown.

   Using solution NMR, we discovered that the monomeric form of RHIM-containing RIPK3, contrary to dominant thinking and predictions, behaves as an intrinsically disordered motif (5). In addition, 15N relaxation, including lifetime line broadening measurements revealed that a 20-residue, flanking stretch that we named the pre-RHIM, participates in the assembly processes. 15N-DEST (Dark-state Exchange Saturation Transfer) experiments and mutagenesis data support that both the RHIM amyloid core mapped by both solid-state NMR and cryo-EM and also the pre-RHIM region are important for amyloid formation. Intriguingly, the pre-RHIM stretch has not been detected in the cited prior structural work and thus has remained unobserved in the resolved amyloid structures. In summary, our findings expand on the structural characterization of RHIM-containing proteins, depicting for the first time their conformations and dynamics in the monomeric, non-assembled state, which are essential to understand not only certain pathways in cell response but also under pathological conditions.

 

Acknowledgements: Grants PID2020-113907RA-I00 and RYC2019-026574-I from MCIN/AEI/10.13039/501100011033 and “ESF Investing in your future” (M. M.), Grant FJC2021-047976-I funded by MCIN/AEI/10.13039/501100011033 and by European Union Next GenerationEU/PRTR (G. A. T.-D.), and Australian Research Council Discovery Project (DP180101275) and National Deuteration Facility Funding NDF7209 and NDF8102 (M. S.) Research Training Program support to N. R. V. from Australian Government. Cofunded by the European Union (ERC, 101042403 – BiFOLDOME). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

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