Mispacking of the F87 sidechain drives aggregation-promoting conformational fluctuations in the subunit interfaces of the transthyretin tetramer

Protein Sci. 2024 Sep;33(9):e5101. doi: 10.1002/pro.5101.

Abstract

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The x-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4% and 7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

Keywords: NMR spectroscopy; conformational dynamics; kinetics; molecular dynamics; protein aggregation; x‐ray crystallography.

MeSH terms

  • Amino Acid Substitution
  • Amyloid Neuropathies, Familial / genetics
  • Amyloid Neuropathies, Familial / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Humans
  • Models, Molecular
  • Prealbumin* / chemistry
  • Prealbumin* / genetics
  • Prealbumin* / metabolism
  • Protein Aggregates
  • Protein Conformation
  • Protein Multimerization

Substances

  • Prealbumin
  • TTR protein, human
  • Protein Aggregates

Supplementary concepts

  • Amyloidosis, Hereditary, Transthyretin-Related