Retinol Binding Protein Complexed with Transthyretin (PDB ID: 1RLB) from Homo sapiens
Created by: Yee Aung
Transthyretin and retinol binding protein complex (PDB ID: 1RLB) functions in transporting thyroxine hormone and retinoic acid simultaneously (1). Transthyretin (TTR) has four identical subunits, each having 127 amino acids. The tertiary structure of TTR is a tetramer. Because of the hydrogen bonding between two beta sheets of each monomer,two monomers form a dimer and there are a total of two dimers in a tetrameric form of one TTR. Four antiparallel strands of β sheets from each monomer contribute to dimer formation. A ligand binding site, a channel with 10 Å in diameter, is formed between two dimers and the two dimers are connected through a symmetric loop (2).These two dimers are connected through β strands by hydrogen bonding (3). Each retinol binding protein (RBP) has the shape similar to a calyx which consists of eight strands of antiparallel β sheets. The β sheets have a short alpha helical segment at the end(4).
The reduced surface area of RBP in solvent, when it is bound to TTR, contributes to the stability of the TTR-RBP protein complex (2).These protein subunit interactions reduce proteins’ surface–to-volume ratio since the bigger the protein, the smaller the surface-to-volume ratio for the protein. Decreased surface-to-volume ratio contributes to the protein’s stability by preventing the hydrophobic residue from interacting with water solvent. When subunits recognize their own subunits or other protein’s subunits, they bind to mutant forms of subunit weakly and this reduces the possibility of genetic translation (5). RBP has surface area of 9356 Ų which can have contact with solvent. However, Because of the interaction with TTR, 736Ų or approximately 8% of RBP is buried in the complex (2).
The molecular weight of RBP is 20054.54 Da and this protein alone can be easily filtered through glomerular filtration in the kidneys (6). The molecular weight of theTTR-RBP complex is 95866.28 Da and it has the isoelectic point of 5.56 (6). The function of the complex formation is important since the RBP-TTR complex could prevent the loss of low molecular weight RBP by the filtration of glomeruli in the kidneys. Preventing the loss of RBP is the same as preventing the loss of the retinoic acid, which is important for physiological and biological processes (4). Therefore, it is known that the structure and formation of RBP and TTR complex contributes to its function (1).
Retinol is important for the biological processes such as cell growth, vision, differentiation and morphogenesis. Since retinol is insoluble and chemically unstable, its necessity for the binding of transport proteins which can solubilize and makes it stable in the binding pocket. RBP also plays in the role of delivering the retinoic acid from mother to the developing fetus (1).
From the observation of x-ray structure, it is known that the retinol binding to RBP stabilizes the complex since there are interactions between the RBP’s ligand entrance loops and TTR (2). Without the retinol, the TTR and RBP cannot form a complex. In the complex, Lys-89, Lys-99 and Trp-67 and Trp-91 of RBP interact with TTR (2).
From crystallographic data of human and chicken TTR-RBP complex, the structure of the RBP-TTR complex is defined as an asymmetric hexamer, meaning two molecules of RBP, with retinoic acid in each RBP, bind to the tetrameric TTR. The complex is an asymmetric hexamer since only two molecules can bind to a TTR tetramer. The binding of RBP molecules, where each RBP molecule is parallel to opposite sides of the x-axis of TTR is close enough to inhibit the binding of other RBP molecules to TTR (2).
The complex formation of RBP with TTR doesn’t affect the binding of T4 to the TTR channel since the binding site channel is involved in the area where there is no interaction between the two kinds of proteins, TTR and RBP (2). It is known that glomerular filtration of RBP can be prevented if the RBP has an uncleavable N terminal signal peptide and N linked glycosylation sites which make RBP heavier. That kind of RBP is called carp RBP and it has the higher molecular weight because of the reason mentioned above (1).
TTR is not the main transport protein for thryoxine (T4) since TBG and albumin plays the major role of thyroid hormone transport. The way it binds is that the negative hydroxyl group of T4 shields the positive residue Lys present in the binding channel of TTR. Triiodothyronine (T3) has lower affinity for TTR compared with T4. The TTR channel has three main parts: hydrophilic center, hydrophobic patch and channel entrance. The hydrophilic center is formed with hydroxyl groups of Ser and Thr and water molecules. Methyl groups of Leu, Thr, Ala and Val form the hydrophobic patch and charged molecules Lys, Glu and His form the entrance of the channel (1).
TTR has evolved by a duplication event in the genetic encoding. Its ancestral molecule protein is 5-hydroxyisourate hydrolase (PDB ID: 2H6U), also known as TTR related protein (TLP) or HIUase. Although they conserve a great amount of amino acids and have similar structures, they performed different functions. TLP from Danio rerio, zebra fish, has a Z score of 18.8 and E value of 3e-13, according to the data given by Dali and Blast search. While the Z score in Dali shows the similarity in tertiary structure, E score shows the similarity in primary structure compared to the TTR protein. Z values greater than 2 and e-scores less than 0.01 indicate that the two proteins show great similarity in their tertiary and primary structure (7, 8).
Although TTR functions as a transport protein for the thyroxine and retinoic acid by forming a complex with the RBP, TLP protein is not a transport protein (9). TLP is also composed of four identical monomers to form a tetramer and two monomers form a dimer through the interaction of β strands as in TTR. Each TLP monomer has eight antiparallel β strands. HIUase is responsible for the catalysis of hydrolyzing 5-hydrosyisourate in the urate degradation process (9).
The difference in structure of TLP and TTR is evident in their binding sites which lead to the functional differences between these two proteins. The active site of the TLP protein is shallower than the ligand binding site of TTR and it is known that TTR adapts itself to transport two thyroid hormones, T3 and T4, which are bound to the channel formed between the two dimer-dimer interphase. TLP does not carry any substrates in its active sites. Two Tyr-116 residues at the bottom of the TLP cavity are substituted by a Thr-119 residue in TTR for the functional transitional change. This change of residues in TLR causes the channel of the ligand binding site to open so that TTR’s transport molecules can fit in. The other three replacements of residues in TLP responsible for TTR function transition are the change of the residues of TLP from His-12 to Lys-15, Asp-50 to Ser-52 and Arg-52 to Glu-54 (9).
In higher organisms, TTR-RBP complex blocks glomerular filtration. Amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA) are the outcomes of the accumulation of the misfolded TTR proteins. FAP is the neurodegenerative lethal disorder caused by systemic deposition of TTR amyloid fibrils in the peripheral nervous system. By preventing the formation of amyloid beta fibrils, TTR prevents Alzheimer’s disease (10). From studies, it is known that the complex protein-protein interaction would prevent TTR from mutation (1). TTR also plays an important role in peripheral and central nervous system physiology by maintaining normal cognitive processes such as ageing and nerve regeneration. The link between TTR and Parkinson’s disease, schizophrenia, and depression is screening techniques (10).