Fig 1. TTR showing all the four subunits.Adapted and modified from PDB file 1ie4

    Transthyretin (formerly called prealbumin) is a compact globular plasma protein. It was named prealbumin because it ran ahead of albumin in electrophoresis gel. Transthyretin(TTR) has monomer molecular weight of approximately 14,000 Da. It is primarily produced in liver and excreted into the plasma (4 ,5 ). TTR mRNA is found in kidney cells (6 ) and is also uniformly distributed in the cytoplasm and epithelial cells of choroids plexus(7 ). TTR has also been found in  pineal gland and retinal pigment ( 8 ) In general plasma Transthyretin levels increase gradually after birth until they reach an adult concentration of 20-40mg/dl, and then decrease after fifth decade.
    Human plasma Transthyretin was crystallized in 1966 from ammonium sulphate solutions by Haupt and Heide( 1 ). It is a tetramer with two alpha beta dimers related by a two fold crystallographic axis. Approximately 45% of the amino acids of the TTR monomer are organized into a sheet of eight beta strands, connected by loops, which result in a classic beta barrel conformation. This unusual high content of beta structure contributed to the extraordinary stability of this molecule. The TTR tetramer is not modified by phosphorylation, glycosylation, or acylation.
    Transthyretin is one of the major three T4 (3,5,3`,5` - tetraiodo-L-thyronine) binding proteins. It binds and transports 15 – 20 % of  thyroxine in serum and up to 80 % of thyroxine in CNS ( 2 ). The thyroxine binding sites are located in a channel (beta barrel) that runs through the centre of the tetramer between two identical dimer units. The ability of TTR to accommodate ligands in a variety of binding modes shows its function as an important transport protein for thyroid molecules. 
    In addition to binding and transporting thyroxine, TTR also transports retinol (Vitamin A) from its main storage site in liver to target cells. The retinol is bound to Retinal Binding Protein (RBP) which, in turn, complexes with transthyretin. RBP would be rapidly eliminated from plasma by glomerular filtration in the kidney if it were not complexed to TTR. Crystal structure of a TTR/RBP complex showed that the TTR tetramer was bound to two molecules of RBP. Rask et al ( 3 ) showed that human RBP exists in two forms only one of which contains retinol.This is the only one which can bind to  TTR.
    Naturally occurring variants of TTR play a  role in amyloidosis, a name given to a group of deposition diseases in which insoluble deposits accumulate in tissues. Familial amyloidotic polyneuropathy (FAP) associated with  TTR variants represents most frequent form of inherited amyloidosis (10 ) where amyloid fibril deposists contain the variant TTR and usually some normal TTR as well. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein-drug complexes gives a detailed analyses of the protein-drug interactions, that stabilize the functional tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR (9 ).

  BACKGROUND :

During evolution, transthyretin synthesis first appeared in the choroid plexus of the stem reptiles, about 300 million years ago. Transthretin synthesis in the liver evolved much later, independently, in birds, eutherians and some marsupial species.Analysis of transthyretin  in 20 different species shows that evolutionary changes of transthyretin predominantly occurred near the N-termini. TTR as already mentioned is a major thyroxine binding protein with 75% of TTR binding to T4(13 ) apart from Thyroxine binding protein and albumin. When thyroxine is not in bound form it partions into lipid memebranes. TTR counteracts this partition and thus there is more thyroxine in TTR bound form than free form. Dewit Goodman first isolated and sequenced Transthyretin. In 1971 Colin Blake determined X-ray structure of human transthyretin. TTR has a very high beta sheet content unlike other three dimentional stuctures of proteins known at that time.Transthyretin variants cause fatal neurological disease called Familial amyloid polyneuropathy (FAP).Amyloid is deposited at certain tissues, kidney, heart and along peripheral nerves.  Previous biophysical studies demonstrated that the mechanism of TTR amyloid fibril formation requires tetramer dissociation to a monomeric conformational intermediate ( 11 , 12 ). This conformational intermediate, generated under conditions simulating the pH of a lysosome (5.5), self-assembles, affording amyloid fibrils (lysosomes are  implicated in amyloid fibril formation) ( 14 , 15 ). 57 human transthyretin variants were analysed which suggested that most mutations in transthyretin are not compatible with its normal metabolism and lead to its deposition as amyloid. Based on cryoelectron microscopy  and fiber diffraction studies, the diameter of  generated amyloid fibril is 130 Å and is made up of four parallel protofilaments  (16 ).

Though TTR contains no carbohydrate it is highly acidic.It was first recognized to bind T4 in 1958 (28 ). Subsequently it was demonstrated that TTR also forms a complex with retinol-binding protein and thus plays a role in the transport of vitamin A (retinol, or trans retinoic acid) (29 , 30 ) and have an important role in nervous system. TTR plays a lesser role in iodothyronine transport in serum relative to that of TBG despite its 20-fold higher concentration. In the presence of normal levels of TBG, wide fluctuations in TTR concentration or its removal from serum  has little influence on the concentration of free T4 ( 31 ). Some Properties and Metabolic Parameters of TTR are as follows ( 32 ):

STRUCTURAL ASPECTS :

        There are two different thyroxine-binding modes. In one mode, thyroxine is positioned in the channel with the phenolic iodines bound in the P2 and P3' pockets of site AC with binding interactions mediated by the water molecules positioned in the P3 pocket not containing the T4 iodine. P3 pocket is formed by ala108,ser117,thr119,leu110 of transthyretin monomer and P2 pocket is formed by ala109,leu110,lys15,leu17 amino acids of transthyretin.The second binding site (B/D) binds the ligand with the phenolic iodines positioned in both P3 and P3' inner pockets. The water molecule found in the middle P2 pocket mediates the T4-binding interactions. Owing to the formation of a 3.07  Å I O interaction to the main-chain carbonyl groups  different binding modes of  T4 is possible (17 ).

   

Fig.4 Downloaded from PDB(1QAB) and modified in rasmol.

   

Fig.5 Plasma Retinol Binding Protein generated from  PDB(1iiu)

The transport of vitamin A to the target tissues is mediated by plasma retinol-binding protein (RBP). Retinol-binding protein is a member of the lipocalin superfamily. It is composed of an eight-stranded beta -barrel and a C-terminal alpha-helix and has a molecular mass of 21 kDa. The retinol is encapsulated by the beta-barrel in the binding cavity in a hand-in-glove-like fit with the ring end of the retinol innermost. Only the solvent accessible part is hydorxyl of retinol( 20 ).

Among the naturally occurring mutations in transthyretin the mutation of Ile-84 to Asn or Ser supports RBP-TTR complex formation. Individuals with this variant of TTR have RBP concentrations substantially lowered in plasma  (20 ) Two molecules of RBP bind to the same TTR dimer at equivalent binding sites. The crystallographic structure at 3.2 Å of the protein-protein complex of human RBP and TTR reveals that RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry. Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than RBP with leu182 and leu183. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.

 

  AMYLOIDOGENIC VARIANTS OF TRANSTHYRETIN

 

  Fig. 6  Transthyretin naturally occurring variant with 1:1 mix of val(green) and met(blue) at position 30 complexed with thyroxine(red) (3,5,3',5'-tetraiodo-l-thyronine) Adapted and modified from PDB file 1ETA