Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-Å resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4°and 49.5°rotations are required to open the N-and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N-and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.The transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal in the body (1, 2). Human serum transferrin (hTF) 4 is synthesized in the liver and secreted into the plasma; it acquires Fe(III) from the gut and delivers it to iron requiring cells by binding to specific transferrin receptors (TFR) on their surface. The entire hTF⅐TFR complex is taken up by receptor-mediated endocytosis culminating in iron release within the endosome (3). Essential to the re-utilization of hTF, iron-free hTF (apo-hTF) remains bound to the TFR at low pH. When the apo-hTF⅐TFR complex is returned to the cell surface, apo-hTF is released to acquire more iron.Strong homologies exist, both between TF family members, and between the two lobes of any given TF (4, 5). Each N-and C-lobe is divided into two subdomains (designated N1 and N2, and C1 and C2) connected by a hinge that gives rise to a deep cleft containing the iron-binding ligands. Iron is coordinated by four highly conserved amino acid residues: an aspartic acid (the sole ligand from the N1-or C1-subdomain), a tyrosine in the hinge at the edge of the N2-or C2-subdomain, a second tyrosine within the N2-or C2-subdomain, and a histidine at the hinge bordering the N1-or C1-subdomain. In addition, the iron atom is bound by two oxygen atoms from the synergistic anion (carbonate), w...

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an antibacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin.Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.

Summary. AL (primary or immunoglobulin light chain) amyloidosis (AL) differs from myeloma per se in that tissue deposits of amyloid are found, typically in association with small numbers of clonal plasma cells producing l light chains, and also in that AL patients typically present with a predominantly dysfunctional organ-system. This constellation of features ± ®brillar deposits comprised of light chains, l light chain predominance, and organ-system tropism and dysfunction ± remains unexplained. Select patients with AL respond to haemopoietic stem cell transplantation (SCT) with clinical improvement and extended survival, particularly those who do not have cardiac involvement. In order to ascertain whether the organ-system tropism of AL was associated with immunoglublin light chain variable region (Ig V L ) germline gene utilization, we attempted to clone, sequence and assign germline donors to the clonal Ig V L genes of 62 AL patients, all of whom were treated with SCT. We succeeded in 39 cases, identifying clonal AL genes derived from donors of the lI (n 10), lII (n 5), lIII (n 6), lVI (n 11) and KI (n 7) subtypes. The majority of the donors (IGLV6S1, DPL5, DPL2, DPL23 and LFVK431) were genes that appear in the expressed repertoire <5% of the time, suggesting an intrinsic propensity to form amyloid under certain conditions. Patients whose clones derived from the lVI IGLV6S1 donor uniformly presented with dominant renal involvement while those with other V l or unknown donors often had dominant cardiac or other organ involvement, particularly patients whose clones derived from the lI DPL2 donor. In addition, both early (<3 months) and overall post-SCT survival were signi®cantly better in lVI IGLV6S1 patients compared to patients with other V l donors. These ®ndings indicate that there are important associations in AL amyloidosis among Ig V L gene utilization, organ-system tropism and post-SCT survival.

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg 2؉ and K ؉ and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS C17S ) in apo form (without ATP bound). EcSPS C17S crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis. Selenium is an essential nutrient that has been linked to many human diseases (27) related to hormone regulation (35, 38), immune response (16, 42), protection from reactive oxygen species (12), and brain function (4). Deficiencies in selenium intake have been implicated in several health disorders over the past two decades, including cancer (5), cardiovascular disease (3), male infertility (43), asthma (39), and viral infections (29). Seleniumcontaining proteins, or selenoproteins, are expressed in many organisms and tissues (26,34,40). Studies have shown that mutations in some of these selenoproteins can lead to decreased activity resulting in abnormal hormone metabolism (8, 9) and rigid-spine muscular dystrophy (30).Selenium has been shown to be incorporated into enzymes/ proteins either as a selenocysteine (7), which occurs at a specific position in the protein sequence and participates in the protein's catalytic action, or as a selenomethionine, which results from random substitution of selenium in place of sulfur in methionine and exerts no effect on the enzymatic activity of the protein (15). Incorporation of selenocysteine, commonly referred to as the 21st amino acid, is mediated by a UGA codon-directed cotranslation involving four genes, selA, selB, selC, and se...