Network of hotspot interactions cluster tau amyloid folds

Abstract

Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformations of β-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We evaluate our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures.

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

“…The distinct aggregation tendencies of WT and GG confirm that the behavior of R3 is influenced not only by the NH6 peptide of the R3 repeat ( 326 GNIHHK 331 ), but also by the NH6 peptide of the preceding repeat ( 264 ENLKHQ 269 ). The influence of the NH6 peptide of R2 repeat ( 295 DNIKHV 300 ) on the conformational properties of R3 was observed previously by Chen et al Therefore, the sequence-dependent modulation provides insights into the conformational diversity offered by NH6 peptides, in line with the role of VQ6 peptides. , The involvement of R1 repeat in aggregation supports its part in promoting fibril formation, as recently demonstrated in our laboratory . 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.…”

“…Protein aggregation has been a prime topic of focus due to its implication in function and pathology. ,,, Identifying the protein misfolding pathways has been quite challenging for IDPs, particularly Tau proteins, which is the primary focus of this work. Cryo-EM structures of Tau fibrils identified since 2017 have a direct correlation to the specific tauopathies, providing confirmation that the amyloid-forming pathways are unique to each type of pathology. ,, This underscores the complexity of these pathways and their association with distinct disease manifestations. While extensive research has been conducted on the amyloidogenic role of the 306 VQIVYK 311 segment in Tau’s R3 repeat, there is comparatively limited understanding regarding the contributions of the other residues within the R3 repeat to the process of amyloid formation. ,, In this work, we probed the break in the sequence grammar of C-terminal NH6 hexapeptides ( 264 ENLKHQ 269 in R1, 295 DNIKHV 300 in R2, 326 GNIHHK 331 in R3, and 358 DNITHV 363 in R4) due to the presence of 326 G at the N-terminus of R3 NH6 peptide, a site predominantly occupied by acidic residues like glutamic acid and aspartic acid.…”

“…Cryo-EM structures of Tau fibrils identified since 2017 have a direct correlation to the specific tauopathies, providing confirmation that the amyloid-forming pathways are unique to each type of pathology. 9,21,66 This underscores the complexity of these pathways and their association with distinct disease manifestations. While extensive research has been conducted on the amyloidogenic role of the 306 VQI-VYK 311 segment in Tau's R3 repeat, there is comparatively limited understanding regarding the contributions of the other residues within the R3 repeat to the process of amyloid formation.…”

Saturation mutagenesis of α-synuclein reveals monomer fold that modulates aggregation

“…To test these mutations for seeding potential, we picked 16 de-enriched and 13 enriched mutants, cloned them upstream of cyan or yellow fluorescent protein (aSyn-CFP or aSyn-YFP), and created high-expressing polyclonal biosensor lines. The literature suggests that these tags do not have any substantial effect on aSyn aggregation ( 31 , 32 ), especially when placed on the C terminus, which remains flexible and disordered in the case of aSyn. We then compared seeding to aSyn(WT) or aSyn(A53T) treated with a range of aSyn fibril concentrations (Fig.…”

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity

“…We next determined how phenylalanine to serine mutations may influence the total SASA of the dimer and the horizontal and vertical hexamers. The F148S, F151S, and F148S_F151S mutations were introduced into the 147 AFSSFN 152 structure using Rosetta and minimized as previously described (Mullapudi, Vaquer-Alicea et al 2022).…”