Human Transthyretin

Human plasma transthyretin at low resolution

Balsa wood model of transthyretin at 6Å resolution. It was impossible to infer the protein main chain or folding at this resolution

Transthyretin (TTR) is a human plasma protein (previously known as prealbumin) from its position relative to human serum albumin on electrophoresis). The renaming reflects its role as a plasma transporter of thyroxine and other thyronines. It also acts as a carrier of the very insoluble retinol through its association with retinol binding protein. So Transthyretin is a dual purpose plasma protein.

Extremely stable towards denaturation, it is a homotetramer with 4 peptide chains of 13.75 Kda approx.. It has by now been very extensively studied and a useful summary can be found on Wikipedia. Today there are a remarkable 361 3D transthyretin structures from various species published in the Protein data bank (PDB). When I started my D.Phil. research a low resolution structure (6 Å ) balsa wood model was available (figure left), but it was impossible to determine the location of the protein subunits The first atomic level structure determination was published in my 1973 Oxford D.Phil thesis and later refined at higher resolution (1).


Solving the 3D structure of transthyretin proved a considerable challenge in the early 70's. The initially available crystals were of a mercury derivative (Hg attached to a single cysteine residue). This caused a small conformational change in the protein so that it was less 'isomorphous' with the native structure. Eventually I solved this by using a reducing agent to remove the mercury atoms from the crystalline protein. A further complication was the 'pseudosymmetry' present in the crystal (no comment!). At that time, protein structures were solved by hanging electron density tracings on acetate sheets behind a partially - reflecting mirror in front of a 'Richards box': a 3D assembly of tensioned wires.

A physical model was then built up using accurately sized wire models (from Cambridge Repetition Engineers) of the amino acids superimposed by reflection within the electron density sheets. An heroic enterprise compared with the sophisticated computer molecular modelling of today. The first structure determination was published in my 1973 Oxford D.Phil thesis (1) and later refined at higher resolution (2).

X=ray diffraction pattern of transthyretin
x-ray diffraction pattern of transthyretin. From dozens sets of data like this and 3 years work (in 1970) it was possible to solve the 3D molecular structure

human plasma transthyretin
View of the tetrameric protein looking down the slot where thyroid hormones bind.

Transthyretin was the second plasma protein 3D structure to be solved (the first was IgG). The stability of transthyretin was immediately clear from the high resolution model. Monomers in the tetrameric structure formed two pairs of continuous ß-pleated sheets and denaturation of the tetramer would probably lead to extensive aggregation.

The ß-pleated sheet structure was fairly novel at the time, but had been observed in some other 3D structures: Concanavalin A and IgG. Up until then, most proteins were assumed to contain large regions of alpha helix. In the view on the left, the large water filled channel between opposing dimers was the obvious place for thyroxine and other ligand binding.

Human plasma transthyretin
A view of the transthyretin dimer separated from the whole 4-subunit molecule. The distinctive association of the subunits in each dimer is their interaction to form two continuous ß-pleated sheets. This tight intersubunit association explains the high stability of the protein.

I soaked transthyretin crystals in a range of ligands, including the thyroid hormone family: Thyroxine (T4) and other iodothyronines (T3 and T2). As anticipated, the thyroid hormone family all bound in the observed channel (centre of image). This channel is very obvious in the low resolution balsa wood model above. I discovered that a number of helminthicides (anti-parasitic drugs) also bound at the thyroid home binding sites. As in the case of HSA, plasma proteins potentially interact with many existing and new drug entities. The result of such binding will usually only emerge in safety and clinical trials!

T4 molecules bound to transthyretin
A view of the intact 4-subunit protein with 2 L-thyroxine molecules occupying the molecule's central channel.

Clinical implications

A much later finding relates to the amyloidogenic property of human transthyretin (TTR) when single point mutations (most commonly valine to methionine) leads to the aggregation of the molecule. Truncation of the N - terminal region of the protein chain seems to prevent native tetramer formation leading to the observed amyloid fibrils. Neuropathy is often a major manifestation of systemic amyloidosis. It is most frequently seen in patients with hereditary transthyretin amyloidosis, but is also present in 20% of patients with systemic immunoglobulin light chain (primary) amyloidosis. Familial amyloid polyneuropathy (FAP) is the most common form of inherited amyloidotic polyneuropathy, with clinical and electrophysiologic findings similar to neuropathies with differing etiologies (e.g., diabetes mellitus). Hereditary amyloidosis is an adult-onset autosomal-dominant disease with varying degrees of penetrance.

It is caused by specific gene mutations, but demonstration that a patient has one such mutation does not confirm the diagnosis of amyloidosis. Diagnosis requires tissue biopsy with demonstration of amyloid deposits either by special histochemical stains or electron microscopy. Transthyretin amyloidosis is treated by liver transplantation, which eliminates the mutated transthyretin from the blood, but for some patients continued amyloid deposition can occur from wild-type (normal) transthyretin [Abstracted from: Merrill D Benson 1, John C Kincaid (2007)] Muscle Nerve 36(4): 411-23

Recent years(2) have seen drugs which interfere with the deposition of misfolded TTR at various stages of the cascade underlying TTR amyloidosis progression. These include TTR tetramer stabilizers (tafamidis, diflunisal, epigallocatechin-3-gallate), TTR silencers (inotersen, patisiran) and fibril disruptors (monoclonal antibodies, doxycycline and tauroursodeoxycholic acid).


References

  1. Structure of prealbumin at 2.5 Å. Blake, C.C.F., Geisow, M.J., Swan, I.D.A., Rerat, C. & Rerat, B.(1974) J Mol Biol 88: 1-12
  2. Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 Å. Blake, C.C., Geisow, M.J., Oatley, S.J., Rerat, B., Rerat, C.(1978) J Mol Biol 121: 339-356.
  3. Emerging therapies in transthyretin amyloidosis – a new wave of hope after years of stagnancy? Maximilian L. Müller,Javed Butler,Bettina Heidecker (2020) European Journal of Heart Failure 22: 39-53