The effect of arrhythmia-associated mutations on the folding, stability and dynamics of the N-terminal domain of the human cardiac ryanodine receptor 2

Vladena Bauerová-Hlinková, Terézia Hromádková, Jacob A. Bauer

Institute of Molecular Biology Slovak Academy of Sciences, Dúbravská cesta 21,

845 51 Bratislava, Slovakia

Ryanodine receptors (RyRs) are large multidomain homotetramers (Mr 3.2 MDa) which are embedded in the membrane of the sarcoplasmic reticulum (SR). Their main physiological function is to release Ca2+ from the SR into the cytoplasm of myocytes, which triggers a cascade of reactions resulting in muscle contraction. Isoform 2 of this receptor is predominantly present in the heart and its dysfunction triggers certain types of arrhythmias (CPVT1, ARVC/D2, syncopes of unknown origin, SIDS). RyR2 dysfunction is often caused by inheritable missense mutations which occur in the gene of this protein. Mutations often affect the oligomeric state of individual domains, their biophysical properties, their dynamics, or their interactions with neighboring domains. These changes alter the opening and closing of the channel.

In our study we focused on the biophysical, biochemical and molecular dynamics characterization of the hRyR2 N-terminal domain (NTD) in its native form and its L433P mutant form, which is associated with CPVT1 and ARVC/D2 [1, 2]. The crystal structure of the hRyR2 NTD in its native form was determined in our laboratory [3]. CD spectroscopy revealed that, although the L433P variant maintains an α+β fold, the L433P mutation increases its α-helical content by 10%. FIDA and SEC analysis showed that the L433P mutation caused an increase in oligomeric formation by the hRyR2 NTD in comparison to the wild-type form. NanoDSF experiments showed that the L433P mutation does not influence the hRyR2 NTD thermal stability. Pilot molecular dynamics experiments suggest that the L433P mutation causes the unwinding of the C-terminal part of the central helix.

1. Monika Seidel, N Lowri Thomas, Alan J Williams, F Anthony Lai, Spyros Zissimopoulos. Cardiovasc Res. 2015 Jan 1;105(1):118-28. doi: 10.1093/cvr/cvu240.

2. Yijun Tang, Xixi Tian, Ruiwu Wang, Michael Fill, S.R. Wayne Chen. Circ Res. 2012 Mar 30; 110(7): 968–977. doi: 10.1161/CIRCRESAHA.111.256560

3. Borko L, Bauerová-Hlinková V, Hostinová E, Gašperík J, Beck K, Lai FA, Zahradníková A, Sevčík J. Acta Crystallogr D Biol Crystallogr. 2014 Nov;70(Pt 11):2897-912. doi: 10.1107/S1399004714020343.

Authors thank to Gabriel Žoldák and Michal Nemergut from Center for Interdisciplinary Biosciences, Pavol Jozef Šafárik University, Košice, Slovakia, for the CD spectra measurements and Eva Kutejová for general support while performing this study. This work was funded by Grant VEGA, no. 2/0081/24.