Covalent Footprinting Mass Spectrometry Maps Early Tau Aggregation Transitions in Microtubule-Binding Domain
Background
Alzheimer's disease and other tauopathies are characterized by the pathological aggregation of tau protein into neurofibrillary tangles, leading to synaptic dysfunction and neuronal loss. While mature fibril structures are increasingly resolved, the initial, dynamic conformational transitions of intrinsically disordered tau that drive aggregation remain poorly understood. This knowledge gap hinders the development of therapeutics targeting early aggregation events, which are critical for preventing disease progression.
Study Design
Researchers utilized diethyl pyrocarbonate (DEPC) covalent footprinting combined with mass spectrometry to capture dynamic conformational changes across full-length human tau 2N4R (441 residues) during a 72 h aggregation period. Recombinant expression of fresh tau 2N4R was used, and optimized DEPC footprinting, along with complementary proteolytic digestion, achieved near-complete (93%) sequence coverage. The primary endpoint was the identification of residue-level protection from solvent exposure, indicating structural transitions during aggregation.
Results
Peptide-level kinetics revealed distinct domain-specific behaviors, with the microtubule-binding domain (MTBD) exhibiting the most pronounced structural transitions. Residue-level analysis precisely mapped these changes onto the fibril core. > Early protection of residues from β4-β6 within the MTBD was identified as a critical early nucleation event in tau aggregation. This was followed by the progressive incorporation of adjacent MTBD strands during fibril maturation. In stark contrast, the N-terminal, proline-rich domain (PRD), and C-terminal regions of tau remained largely solvent-exposed throughout the entire 72 h aggregation process, suggesting they do not participate in the core fibril formation at these early stages. These findings provide unprecedented detail on the dynamic structural evolution of tau during its pathological aggregation.
Key Findings
- Covalent footprinting achieved 93% sequence coverage of full-length human
tau 2N4Rduring aggregation. - The
microtubule-binding domain (MTBD)showed the most significant structural transitions during tau aggregation. - Early protection of residues from
β4-β6within theMTBDwas identified as an initial nucleation event. - N-terminal, proline-rich, and C-terminal regions of tau remained largely solvent-exposed throughout 72 h aggregation.
Why It Matters
This study offers a groundbreaking methodology for observing the elusive early conformational changes in tau, which are crucial for understanding tauopathy pathogenesis. By pinpointing specific residues and domains involved in early nucleation (e.g., β4-β6 in MTBD), this research opens new avenues for rational drug design targeting these initial aggregation steps. It provides a complementary tool to existing structural techniques, which often focus on mature aggregates, allowing for the identification of novel therapeutic targets that could prevent or slow tau aggregation before extensive pathology develops. This could lead to more effective interventions for Alzheimer's disease and related neurodegenerative conditions.
tau
tauopathy
alzheimers-disease
protein-aggregation
mass-spectrometry
in-vitro