Thermodynamic Stability and Aggregation Kinetics of EF Helix and EF Loop Variants of Transthyretin.

Abstract

Misfolding and aggregation of transthyretin (TTR) are linked to amyloid disease. Amyloidosis occurs when the TTR homotetramer dissociates into aggregation-prone monomers that self-assemble into amyloid. In familial transthyretin amyloidosis, hereditary amino acid substitutions destabilize TTR and promote aggregation. In this work, we used 19F nuclear magnetic resonance (NMR) to determine the effect of mutations in the EF helix (Y78F, K80D, K80E, and A81T) and EF loop (G83R and I84S) on the aggregation kinetics and stability of the TTR tetramer and monomer. The EF region acts as a scaffold that stabilizes interactions in both the strong and weak dimer interfaces of the tetramer and is the site of a cluster of pathogenic mutations. K80D and K80E are non-natural mutants that destabilize the EF helix and yield an equilibrium mixture of tetramer and monomer at neutral pH, providing a unique opportunity to determine the thermodynamic parameters for tetramer assembly under nondenaturing conditions. Of the pathogenic mutants studied, only A81T formed appreciable monomer at neutral pH. Real-time 19F NMR measurements showed that the pathogenic Y78F mutation accelerates aggregation by destabilizing both the tetrameric and monomeric species. The pathogenic mutations A81T, G83R, and I84S destabilize the monomer and increase its aggregation rate by disrupting a Schellman helix C-capping motif. These studies provide new insights into the mechanism by which relatively subtle mutations that affect tetramer or monomer stability promote entry of TTR into the dissociation-aggregation pathway.