Human Serum Transferrin (Apo-form) (PDB ID: 2HAU)
Created by James Ramadan
Human Serum Transferrin (hTF) is a blood plasma glycoprotein produced in the liver and is the major iron binding protein in the vertebrate serum. The main role of hTF is to carry ferric iron (Fe3+) from the intestine, reticuloendothelial system, and liver parenchymal cells to all proliferating cells in the body [1]. These cells require iron because they are full of iron-containing enzymes and proteins, often containing heme prosthetic groups, which participate in many biological oxidations and in transport. Some examples of these iron-containing proteins include hemoglobin, cytochrome, and catalase. Imbalanced transferrin levels can cause seriously negative health consequences. Increased hTF levels often lead to iron deficiency anemia, while decreased hTF levels can lead to iron overload diseases and protein malnutrition. An absence of transferrin in the body creates a rare genetic disorder known as atransferrinemia; a condition characterized by anemia and hemosiderosis in the heart and liver that leads to many complications including heart failure [18].
hTF has a unique structure and special properties which drive its function. Transferrin has a molecular weight of around 80 kDa and its isoelectric point is around 6 but the exact value depends on the iron and sialic acid content of the molecule [4]. It consists of a polypeptide chain containing 676 amino acids, and is composed of 31% alpha helices (29 helices; 213 residues) and 19% beta sheets (29 strands; 122 residues) [10]. Two different crystal structures have been determined for apo-hTF depending on the glycosylation pattern. Although essentially identical, these two structures differ in the number of citrate molecules they can bind [12].
The polypeptide chain of hTF consists of two homologous domains, the N and C- termini, which can each bind one iron molecule and are connected by a linker peptide. Each of the domains is further separated into two subdomains: the N1-(1-92 and 247-331) and C1-(339-425 and 573-679) are each composed of two discontinuous sections of the polypeptide chain, whereas the N2-(93-246) and C2-(426-572) subdomains are each composed of a single region of continuous polypeptide. The two domains, the N- and C- termini, show some interesting differences including the presence of both asparagine-linked glycan moeties in the C-terminal domain at positions 413 and 610, and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). Despite these differences, the homologous domains have 40% identical residues to one another when aligned by inserting gaps at appropriate positions, and there is no difference in the in vivo behavior of the sites with respect to iron uptake and delivery to the cells. It has been suggested that the ancestral protein of hTF had only one iron binding site, and that the evolutionary advantage of the doubled structure is related to the reduction of losses on glomerular filtration[10].
hTF contains 2 specific high affinity Fe (III) binding sites. The amino acids which bind ferric iron to the hTF are identical for both domains; they are two tyrosines, one histidine, and one aspartic acid [5]. At the N-terminus, the amino acids participating in binding iron are Asp-63, Tyr-95, Tyr-188, and His-249, and at the C-terminus they are Asp-392, Tyr-426, Tyr-517, and His-585[12]. hTF changes its conformation depending on whether iron is bound. When iron is not bound, hTF is known as apo-human serum transferrin (apo-hTF) and is in an open conformation, whereas when iron is bound, hTF exists in a closed conformation. The closed conformation of the N-terminus is shown (PDB ID: 1D3K), noting the active amino acids involved. In order for the iron ion to bind to hTF an anion is required, preferably bicarbonate [17]. The binding and release of iron by apo-hTF result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge [11]. A triad of amino acids, namely Lys-534, Arg-632, and Asp-634, has been implicated in the conformational change in the C-lobe during iron release. A cooperative effect between the two lobes leads to further conformation changes in hTF[12].
After loading these two sites with iron, the hTF protein binds to a transferrin receptor on the surface of a cell, and as a result is transported into the cell as a vesicle via endocytosis. The transferrin receptor (TFR) is a homodimeric membrane protein composed of an endodomain anchored in the plasma membrane and an ectodomain directed toward the biological fluids which interacts with the two iron-loaded transferrins [11]. When hTF is iron-loaded, the TFR binds to Pro142-Pro145 of the N2 subdomain and Lys365-Asp372 of the C1 subdomain. The transferrin receptor-transferrin complex enters the cell and releases its iron ions due a decrease in pH caused by hydrogen ion pumps [2]. The affinity of hTF for Fe (III) is extremely high at pH 7.4 (blood pH) but decreases progressively with decreasing pH below neutrality [3]. After releasing the iron, hTF and its receptor are then transported back to the cell surface so that hTF can bind more iron [2].
In addition to iron, hTF can bind citrate, glycerol and aluminum. hTF binds various amounts of citrate depending on the glycosylation pattern. Citrate is a chelator that enhances apo-hTF's affinity for binding iron [12]. hTF can also bind glycerol molecules. When determining apo-hTF's crystal structure, glycerol was used primarily as a stabilizing agent [12]; however, hTF's affinity for glycerol may have some biological relevance. It has been demonstrated that the addition of very small amounts of transferrin to rat adipocytes in vitro resulted in a dose-dependent increase in lipolysis (glycerol release) [13]. Finally, hTF can bind nonferric metallic elements such as aluminum, and it is considered as a possible vehicle for aluminum from bloodstream to cytosol. Aluminum is involved in dialysis dementia, is a neurotoxicant, and causes nephritic disorders and pulmonary fibrosis. Aluminum transport in humans supposedly follows the iron transport pathway and occurs via the aluminum-loaded human transferrin interaction with the transferrin receptors. Transferrin appears to be the predominant aluminum carrier in biological fluids and across the blood brain barrier [11].
Human serum transferrin is part of a bigger family referred to as the transferrin family and has many similarities in structure and function to other members of its family. Lactoferrin (PDB ID: 1DTZ), also a member of this family, is a globular glycoprotein with a molecular weight of around 80 kDa and an isoelectric point of 8.7 [6]. Like hTF, lactoferrin transfers iron to cells and controls the level of free iron in the blood and external secretions; however unlike hTF, lactoferrin is primarily found in secretory fluids, such as milk, saliva, and tears, and not in blood. Lactoferrin is also thought to play a role in the immune system; it has antimicrobial activity and is part of the innate defense, mainly at mucoses [7]. Despite the fact lactoferrin has about a 61% sequence similarity (Z score 40.6) to hTF [14] while in apo-form, lactoferrin's affinity for iron is 300 times higher than that of transferrin and this affinity increases in weakly acidic medium. This difference in affinity for iron is important because when the pH of tissues decreases during inflammations, due to the accumulation of lactic and other acids, hTF can transfer its iron to lactoferrin in order for lactoferrin to perform its proper immune functions [8]. Structurally, the difference in affinity is thought to be related to the fact the linker peptide in lactotransferrin is helical while the linker peptide is in an extended (unstructured) conformation in hTF. The rigid helical peptide link is thought to increase cooperativity between lobes in lactoferrin [16]. Another glycoprotein in the transferrin family with around 54% sequence similarity (Z score 41.3) to hTF is duck ovotransferrin (PDB ID: 1AOV) [14]. Ovotransferrin is 686 residues long and also transfers iron to cells, but in egg white albumen. Structurally, ovotransferrin differs from hTF in its glycosylation pattern [9]. In contrast to family members human lactoferrin and duck ovotransferrin, both of hTF's globular lobes are almost equally open: 59.4 degrees and 49.5 degrees are required to open the N- and C-lobes, respectively [5].
In conclusion, it is human serum transferrin's unique structure that allows it to bind iron and properly regulate free blood iron levels. hTF can bind up to two iron in the blood and transport them to the cytosol of cells via endocytosis after forming a transferrin-transferrin receptor complex. Various molecules, such as citrate, enhance irons ability to bind to hTF while some, such as carbonate or a similar natured anion, are required for hTF to bind iron. Because hTF's affinity for iron is pH dependent, the cell can mediate when hTF should bind iron and when it should release it. Without this regulation of iron, human life would not be possible.