Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins

Mason, Anne B.; Halbrooks, Peter J.; Larouche, Julia R.; Briggs, Sara K.; Moffett, Marque L.; Ramsey, Jon E.; Connolly, Susan A.; Smith, Valerie C.; MacGillivray, Ross T. A.

doi:10.1016/j.pep.2004.04.013

Cited by 40 publications

(96 citation statements)

References 58 publications

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“…Previous studies demonstrated that this mutant (aTf) completely lacks the ability to bind iron in either lobe (18). Furthermore, this construct has a mass that is significantly different from that of the wild-type hTf (76.8 vs. 79.7 kDa) due to the absence of the glycosylation and the presence of the His-tag, allowing distinction between ðFe 2 TfÞ 2 · TfR and a putative ðaTfÞ 2 · TfR complex.…”

Section: Resultsmentioning

confidence: 97%

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

Leverence

Mason

Kaltashov

2010

Proc. Natl. Acad. Sci. U.S.A.

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The primary route of iron acquisition in vertebrates is the transferrin receptor (TfR) mediated endocytotic pathway, which provides cellular entry to the metal transporter serum transferrin (Tf). Despite extensive research efforts, complete understanding of Tf-TfR interaction mechanism is still lacking owing to the complexity of this system. Electrospray ionization mass spectrometry (ESI MS) is used in this study to monitor the protein/receptor interaction and demonstrate the ability of metal-free Tf to associate with TfR at neutral pH. A set of Tf variants is used in a series of competition and displacement experiments to bracket TfR affinity of apo-Tf at neutral pH (0.2-0.6 μM). Consistent with current models of endosomal iron release from Tf, acidification of the protein solution results in a dramatic change of binding preferences, with apo-Tf becoming a preferred receptor binder. Contrary to the current models implying that the apo-Tf/TfR complex dissociates almost immediately upon exposure to the neutral environment at the cell surface, our data indicate that this complex remains intact. Iron-loaded Tf displaces apo-Tf from TfR, making it available for the next cycle of iron binding, transport and delivery to tissues. However, apo-Tf may still interfere with the cellular uptake of engineered Tf molecules whose TfR affinity is affected by various modifications (e.g., conjugation to cytotoxic molecules). This work also highlights the great potential of ESI MS as a tool capable of providing precise details of complex protein-receptor interactions under conditions that closely mimic the environment in which these encounters occur in physiological systems.metalloprotein | protein interaction

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Section: Resultsmentioning

confidence: 97%

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

Leverence

Mason

Kaltashov

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Recombinant nonglycosylated monoferric hTF, designated Fe N hTF, contains mutations preventing iron acquisition by the C lobe (Y426F/Y517F) and glycosylation (N413D and N611D) and is produced in a BHK cell expression system (35). Likewise, the glycosylated ectodomain of the TFR (residues 121-760) is produced and secreted into the tissue culture medium of transfected BHK cells (29).…”

Section: Methodsmentioning

confidence: 99%

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

Eckenroth

Steere

Chasteen

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe N hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

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“…Following a 4-h wash-in period this media was discarded and replaced with 250 ml of the same medium for an additional 48 h of incubation. The recombinant hTF-NG was purified from the medium as described in detail (30). In two production runs between 8 and 16 mg of SeMet containing N-His hTF-NG was produced, of which approximately half was recovered.…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

Wally

Halbrooks

Vonrhein

et al. 2006

Journal of Biological Chemistry

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259

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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...

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Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins

Cited by 40 publications

References 58 publications

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

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