Serine-404 Phosphorylation and the R406W Modification in Tau Stabilize the cis-Proline Amide Bond, via Phosphoserine-Proline C–H/O and Proline-Aromatic C–H/π Interactions

Tau misfolding, oligomerization, and aggregation are central to the pathology of Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), frontotemporal dementia, and other tauopathies. Increased phosphorylation of tau is associated with conformational changes that are not fully understood. Moreover, tau oligomerization and aggregation are associated with proline cis-trans isomerism, with the phosphorylation-dependent prolyl isomerase Pin1 reducing tau hyperphosphorylation and aggregation. The FTDP-17 tau mutation R406W is frequently used in animal models of Alzheimer's disease, due to earlier onset of the AD phenotype. Despite its extensive application, the mechanisms by which tau-R406W leads to enhanced aggregation and neurotoxicity are poorly understood. Peptides derived from the tau C-terminal domain were examined by NMR spectroscopy as a function of residue 406 identity (Arg versus Trp) and Ser404 phosphorylation state. The R406W modification led to an increased population of Pro405 cis amide bond, which is stabilized by cis-proline-aromatic C–H/π interactions. Ser404 phosphorylation also resulted in an increase in cis amide bond, via a proposed C–H/O interaction between the Pro Hα and the phosphate that stabilizes the cis conformation. An analogous C–H/O interaction was observed in Glu-cis-Pro sequences in the PDB, and is proposed to be the basis of the increased propensity for cis amide bonds in Glu-Pro sequences. The higher activation barriers for proline cis-trans isomerization observed at pSer-Pro and pThr-Pro sequences are proposed to be due to both (a) an intraresidue phosphate-amide bond that stabilizes the trans-proline conformation and (b) the cis-stabilizing proline-phosphate C–H/O interaction identified herein. The combination of both pSer404 and R406W resulted in a further increase in the population of cis amide bond. In contrast to expectations, the R406W modification led to increased dephosphorylation of either pSer404 or pSer409 by PP2A, and had no effect on phosphorylation of Ser404 by cdk5, suggesting that R406W does not inherently increase Ser404 phosphorylation via changes in the actions of these enzymes. Modestly increased phosphorylation of Ser404 was observed by GSK-3β in tau R406W. Collectively, these data suggest a potential role for conformational change to a cis amide bond at Pro405, via Ser404 phosphorylation and/or R406W modification, as a possible mechanism involved in protein misfolding in AD, CTE, and FTDP-17. Alternatively, both Ser404 phosphorylation and the R406W modification lead to increased order, including induced turn formation, in both the trans-proline and cis-proline conformations.