Scientists from Scripps Research have uncovered new structures of transthyretin, a protein that moves important hormones like thyroxine through blood and spinal fluid. Misfolded versions of the protein form dangerous clumps in the heart and along nerves which trigger the development of transthyretin amyloidosis (ATTR), a progressive and fatal disease. A deeper understanding of the protein structure could contribute to the development of new drugs for ATTR which affects up to a quarter of all men over the age of 80 to varying degrees.
Their findings were published in Nature Structural & Molecular Biology in a paper titled, “The conformational landscape of human transthyretin revealed by cryo-EM.” In it, the scientists explained that by resolving the structures, they could demonstrate how its three-dimensional asymmetry may contribute to its instability.
Jeffery Kelly, PhD, a professor of chemistry at Scripps and a co-senior author of the study, noted that “the new structures reveal differences in two thyroid hormone binding sites previously thought to be identical, and help explain why a drug binding to one site changes the ability of drugs to bind the opposing site.” Furthermore, “we’ve unveiled a molecular complexity that has been hidden from researchers for decades, which enables us to design better medicines to stabilize transthyretin,” Gabriel Lander, PhD, co-senior author and professor in the Scripps’ department of integrative structural and computation biology, added.
To determine the three-dimensional structure of the proteins, the researchers used cryo-electron microscopy (cryo-EM) with some modifications to account for challenges with protein suspension. Traditional cryo-EM flash-freezes proteins to catch their natural structures and then suspends them in a liquid. Small proteins like transthyretin tend to get stuck at the air-liquid boundary rather than remaining fully submerged in the liquid. This affects both the proteins’ structural stability and makes it difficult to get details about their structures.
To overcome this challenge, the team developed a thin graphene-coated grid to which the transthyretin molecules could naturally adhere. Then, they rapidly plunged this grid into liquid ethane to freeze the sample. This process trapped the transthyretin molecules in place on the graphene surface and preserved their natural conformations to mimic how they would appear when moving through the body’s fluids.
This approach is based on work done in the laboratory of Nieng Yan, PhD, at Princeton University. Benjamin Basanta, PhD, a former research associate in the Lander Lab and first author of the new paper, explained, “Getting the surface chemistry right is crucial for this type of study. With small proteins like transthyretin, creating a high-quality sample is just the beginning; analyzing the data is also part of the challenge.”
When the team analyzed the transthyretin on the grid, they found that the protein forms asymmetric structures with two differently shaped binding pockets. Based on the more than 200 crystal structures that had been solved in the past, these binding sites were assumed to be identical. However, the researchers showed that this variation was because the transthyretin complex moves between two different states—like a molecular version of “breathing,” according to Lander. This asymmetry in the native structure of transthyretin also presented a possible hypothesis for how the process of dissociation and misfolding occurs and leads to the clumping associated with ATTR.
As part of the study, the team tested the effects of an existing ATTR drug called tafamidis, which was also developed by Scripps researchers in 2019. Two formulations of the drug—Vyndaqel (tafamidis meglumine) and Vyndamax (tafamidis)—are the first FDA-approved treatments for transthyretin amyloid cardiomyopathy (ATTR-CM). When the drug was applied to one or both of the transthyretin binding sites in the current study, the researchers found that treatment stabilized the molecule and minimized its movements.
Now the Scripps scientists aim to study how transthyretin’s shifting structure and its stabilization relate to ATTR, and how drugs that target transthyretin could treat the disease. They also believe that their graphene grid could be helpful for determining the structures of other small and unstable proteins like the amyloid-beta peptide that builds up in the brain in cases of Alzheimer’s disease.
“The methodologies we’ve developed have opened new avenues of treatment that could one day protect patients from not just TTR amyloidosis, but other amyloid diseases as well,” Lander said.
