The biological and chemical implications of ligand binding to the amyloidogenic protein transthyretin

Transthyretin (TTR) is a homotetrameric protein that transports the holo-retinol binding protein and the small molecule hormone thyroxine in the blood and cerebral spinal fluid. Both wild-type TTR and over 100 variants of TTR have been found deposited as fibrillar (amyloid) and/or other amorphous aggregates in cardiac tissue, peripheral nerves, and the central nervous system in a process genetically and biochemically linked to human disease. Incorporation of traps-suppressor subunits into the TTR tetramer has been demonstrated to ameliorate TTR-related amyloid diseases in compound heterozygotes by kinetically stabilizing the native TTR tetramer, slowing the rate of tetramer dissociation, the rate-limiting step of TTR aggregation. The work presented herein demonstrates that small molecule binding to the two thyroxine binding sites of the TTR tetramer similarly increases the kinetic barrier for tetramer dissociation, kinetically stabilizing the native tetramer and preventing aggregation. This kinetic stabilization is demonstrated under both denaturing conditions (e.g. high urea concentrations) and physiological conditions. Using both protein engineering techniques and mathematical modeling, the sufficiency of binding a single inhibitor for the inhibition of tetramer dissociation is demonstrated, suggesting that inhibitors with strong negatively cooperative binding between the two thyroxine binding sites will be the most effective inhibitors of TTR tetramer dissociation. Small molecule binding to the TTR tetramer is also demonstrated to be a potential cause of the specific tissue deposition of certain TTR variants in the central nervous system by stabilizing highly unstable TTR tetramers, which would normally be degraded by the cellular quality control system through the secretory pathway. This work represents the foundation for the use of small molecule kinetic stabilizers for the treatment of TTR-related amyloid diseases and potentially a number of other protein aggregation diseases.