Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti‐tau antibody AT8

Abstract

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Identification of the Tau phosphorylation pattern that drives its aggregation

“…Because no G207 Tau mutation has ever been described, we wondered whether a PTM might destabilize the turn-like structure in a similar manner. An obvious candidate is the phosphorylation of the nearby Ser208, recently added to the AT8 epitope on top of the phosphorylated Ser202/Thr205 residues (15). The crystal structure of a triply phosphorylated Tau peptide in complex with the AT8 Fab shows the peptide in an extended conformation compatible with a model whereby it would interfere with the turn-like structure in a similar way as the G207V mutation (SI Appendix, Fig.…”

“…Indeed, Tau phosphorylation at the three positions, Ser202/Thr205/Ser208, while not at Ser262, is sufficient to induce aggregation without the addition of any exogenous aggregation inducer. The former three sites correspond to the AT8 epitope, as recently redefined on the basis of the AT8 Fab crystal structure in complex with the triply phosphorylated peptide (15), and hence suggest the antibody, clinically used to stage the disease progression (10), indeed immunostains a Tau species that can aggregate without any other factors. Whereas the quest for aggregation inhibitors has been mainly performed with heparin as an external aggregation inducer (46)(47)(48)(49), our present description of a phosphorylation-driven Tau aggregation assay opens up new avenues and should allow retesting of the previously defined classes of molecules or the discovery of potentially more specific molecules that counteract the aggregation of phosphorylated Tau.…”

“…Notably, Braak staging, whereby the cognitive decline of deceased patients is correlated with the extent of the lesions in their brain, has been standardized on the basis of immunochemistry with the AT8 antibody (10). The recent finding that the optimal epitope of this clinical antibody is not limited to the double phosphorylation at Ser202/Thr205 of Tau but also can include the modification of the Ser208 residue was quite unexpected (15). Whereas the former 2P-AT8 epitope is characterized by a turn-like structure (14), we show here that this structure can be disrupted either by mutating the Gly207 to Val or by phosphorylation of the Ser208 (Fig.…”

“…Homonuclear nuclear Overhauser (NOESY) and total correlation (TOCSY) spectra were acquired at 293 K with standard pulse programs on 2-mM peptide samples in a phosphate buffer (50 mM phosphate buffer at pH 6.4, 25 mM NaCl, 2.5 mM EDTA, and 5% D 2 O). 1 H, 15 N heteronuclear single quantum coherence (HSQC) spectra were acquired at natural abundance, with 128 scans per increment and 2,048 × 256 complex points in the direct and indirect dimensions, respectively. For protein NMR experiments, Tau proteins were dissolved at a concentration of 200-300 μM in NMR sample buffer (50 mM phosphate buffer at pH 6.4, 25 mM NaCl, 2.5 mM EDTA, 1 mM DTT, and 10% D 2 O).…”

“…A combined NMR and molecular dynamics study showed that this phosphopeptide adopts a particular turnlike structure in solution (14). Only very recently has the crystal structure of its fragment antigen body (Fab) with different phosphorylated peptides suggested that a third phosphorylation event at position Ser208 might lead to an epitope with an even better affinity (15). However, whether phosphorylation is a driver of aggregation remains unclear, and even more so the precise definition of the phosphopattern or phosphopatterns that can cause the aggregation of Tau.…”