NMR Relaxation Experiments Probe Monomer–Fibril Interaction and Identify Critical Interacting Residues Responsible for Distinct Tau Fibril Morphologies

Abstract

Tau aggregation is governed by secondary processes, a major pathological pathway for tau protein fibril propagation, yet its molecular mechanism remains unknown. This work uses saturation transfer and lifetime line-broadening experiments to identify the critical residues involved in these secondary processes. Distinct residue-specific NMR relaxation parameters were obtained for the truncated three repeat tau construct (K19) in equilibrium with structurally different, self-aggregated (saK19) or heparin-induced (hK19) fibrils. The interacting residues are restricted to R3 repeat for hK19 and to R3, R4, and R′ repeats for saK19 fibrils. Furthermore, the relaxation profiles of tau monomers in equilibrium with the structurally comparable, in vitro pathological fibrils (tauAD and tauCTE) were similar but distinct from hK19 or saK19 fibrils. Thus, residue-specific relaxation identifies the important residues involved in the binding of monomers to the fibrils. The relaxation profile of the monomers in equilibrium with the NMR invisible fibril seeds potentially distinguishes the distinct structures of tau fibrils.

Modulation of Primary and Secondary Processes in Tau Fibril Formation by Salt-Induced Dynamics

“… ,, The relaxation studies on the C322A construct confirm the absence of mutual interactions of the two motifs in the monomeric form. In our recent study, we observed that Δ R 2 profiles of 3R tau in the presence of tauAD and tauCTE seeds showed similar Δ R 2 profiles for these two motifs as observed in this study . Thus, based on the Cryo-EM structure and our NMR relaxation studies, we propose that the modulation dynamics of 321 KCGS and 364 PGGGN motifs will shape the final structure of AD/CTE filaments.…”

Role of G326 in Determining the Aggregation Propensity of R3 Tau Repeat: Insights from Studies on R1R3 Tau Construct

“…The participation of the R1 repeat in templated aggregation was also observed in recent studies of K19 Tau (R1R3R4TH6) constructs from our laboratory. 50 The inferences from NMR aggregation studies were corroborated by using ThT fluorescence measurements (Figure S6) under the same NMR experimental conditions. GG construct shows accelerated kinetics with a half-time (t 1/2 ) of 12.5 days compared to WT (t 18 1/2 days) thereby showing that the NMR studies probed aggregation at the early stages.…”

“…53,66 The involvement of R1 repeat in aggregation supports its part in promoting fibril formation, as recently demonstrated in our laboratory. 50 Results from NMR experiments that probed backbone conformations, such as average amide chemical shift differences and secondary structure propensities, reveal that the residues of the AIM are perturbed due to the G326E mutation. Further, the changes in backbone conformation at the G326E mutation site significantly impact the local dynamics at the AIM, as evidenced by the pronounced differences in relaxation rates of 317 KVTSKCGS 324 segment.…”

“…Solution NMR 47,48 has been the powerhouse experimental tool for studying monomeric properties of IDPs, particularly the Tau protein, as it offers insights into the residue-specific perturbations. 5,11,31,36,49,50 For our study, we employed two R1R3 constructs: the R1R3 wild-type (WT) and the R1R3-G326E mutant (EE) (Figure 1C). The conformational changes occurring on G326E mutation are probed through average 1 H− 15 N chemical shift differences and 13 C secondary chemical shift differences.…”