Revealing the conformational landscape of tau P → L mutants in R23 tau macromolecule: A pathway to targeted...

Misfolding of the microtubule-associated protein tau is central to the pathology of several neurodegenerative disorders, including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy. Although proline-to-leucine (P → L) substitutions in tau are known to accelerate aggregation, the molecular mechanisms underlying this effect-and potential strategies to counter it-remain incompletely understood. Here, we use replica-exchange molecular dynamics (REMD) to investigate how single (P301L, P332L) and double (P2L) P → L mutations in tau's R23 fragment reshape its conformational landscape. Our simulations reveal that single mutations substantially elevate β-sheet content (by up to 17% compared to wild-type) and increase aggregation propensity. Paradoxically, the double mutant reduces β-sheet formation and enhances α-helical structure, effectively curtailing aggregation. Contact map and hydrogen-bond analysis confirm that introducing the second P → L substitution reorganizes intrachain interactions in a way that mitigates misfolding. These findings advance our fundamental understanding of tau aggregation and suggest a counter-intuitive therapeutic concept: engineering an additional mutation may stabilize tau more effectively than merely suppressing a pathogenic mutation. We propose integrative next steps-virtual screens, AI clustering, and ssNMR-to guide translation.