Residual Dipolar-Coupling-Based Conformational Comparison of Noncovalent Ubiquitin Homodimer with Covalently Linked Diubiquitin.

Although the conformation of the polymer chain of Ubiquitin (Ub) mainly depends on the type of isopeptide linkage connecting two Ub molecules, the non-covalent (noncovalent) interaction between two Ub molecules within the chain could also tune their conformational preference. Here, we studied the conformation of noncovalently formed Ub dimers in solution using residual dipolar couplings (RDCs). Comparing the RDC derived alignment tensor of the noncovalently formed dimer with the two most abundant (K11 and K48) covalent linked Ub dimers revealed that the conformation of K11 linked and noncovalent Ub dimers were similar. Between the various NMR and crystal structures of K11 linked Ub dimers, RDC tensor analysis showed that the structure of K11 linked dimer crystalized at neutral pH is similar to noncovalent dimer. Analogous to the experimental study, the comparison of predicted order matrix of various covalent Ub dimers with that of the experimentally determined order matrix of noncovalent Ub dimer also suggests that the conformation of K11 linked dimers crystalized at neutral pH is similar to the noncovalent dimer.

Zn2+ Interrupts R4-R3 Association Leading to Accelerated Aggregation of Tau Protein.

Direct binding of divalent metal ion, especially Zn2+ , have been shown to increase the rate of tau aggregation and enhance tau toxicity in cells. Hence, understanding the molecular basis of the Zn2+ -accelerated tau aggregation can potentially determine the molecular interactions modulating tau aggregation. Herein, we show that Zn2+ coordinates through the cysteine in R3 repeat and significantly accelerates the aggregation rate of the three repeat tau constructs (K19) but that the coordination is incapable of increasing the aggregation rate of the 20 amino acid peptide derived from the R3 repeat (R3) of tau. The NMR characterization of the binding of Zn2+ to K19, together with the aggregation studies with K19, R3 and R4 peptides, reveal the presence of an aggregation-inhibitory interaction between the R3 and R4 repeat of K19. Our data show that binding of Zn2+ to R3 repeat of tau, weaken the aggregation-inhibiting influence between R3 and R4 repeats, leading to faster aggregation of tau protein.

Direct Observation of Aggregation-Induced Backbone Conformational Changes in Tau Peptides.

In tau proteins, the hexapeptides in the R2 and R3 repeats are known to initiate tau fibril formation, which causes a class of neurodegenerative diseases called the taupathies. We show that in R3, in addition to the presence of the hexapeptides, the correct turn conformation upstream to it is also essential for producing prion-like fibrils that are capable of propagation. A time-dependent NMR aggregation assay of a slow fibril forming R3-S316P peptide revealed a trans to cis equilibrium shift in the peptide-bond conformation preceding P316 during the growth phase of the aggregation process. S316 was identified as the key residue in the turn that confers templating capacity on R3 fibrils to accelerate the aggregation of the R3-S316P peptide. These results on the specific interactions and conformational changes responsible for tau aggregation could prove useful for developing an efficient therapeutic intervention in Alzheimer's disease.