title: Identification of a kinetically significant anion binding (KISAB) site in the N-lobe of human serum transferrin.
abstract: Human serum transferrin (hTF) binds two ferric iron ions which are delivered to cells in a transferrin receptor (TFR) mediated process. Critical to the delivery of iron to cells is the binding of hTF to the TFR and the efficient release of iron orchestrated by the interaction. Within the endosome, iron release from hTF is also aided by lower pH, the presence of anions, and a chelator yet to be identified. We have recently shown that three of the four residues comprising a loop in the N-lobe (Pro142, Lys144, and Pro145) are critical to the high-affinity interaction of hTF with the TFR. In contrast, Arg143 in this loop does not participate in the binding isotherm. In the current study, the kinetics of iron release from alanine mutants of each of these four residues (placed into both diferric and monoferric N-lobe backgrounds) have been determined +/- the TFR. The R143A mutation greatly retards the rate of iron release from the N-lobe in the absence of the TFR but has considerably less of an effect in its presence. Our data definitively show that Arg143 serves as a kinetically significant anion binding (KISAB) site that is, by definition, sensitive to salt concentration and critical to the conformational change necessary to induce iron release from the N-lobe of hTF (in the absence of the TFR). This is the first identification of an authentic KISAB site in the N-lobe of hTF. The effect of the single R143A mutation on the kinetic profile of iron release provides a dramatic illustration of the dynamic nature of hTF.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, Vermont 05405, USA.
authors.forename: Shaina L
authors.initials: SL
authors.lastname: Byrne
authors.forename: Ashley N
authors.initials: AN
authors.lastname: Steere
authors.forename: N Dennis
authors.initials: ND
authors.lastname: Chasteen
authors.forename: Anne B
authors.initials: AB
authors.lastname: Mason
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry; D001665:Binding Sites; D002478:Cells, Cultured; D006224:Cricetinae; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000838:Anions; D014168:Transferrin
references.citation: J Biol Chem. 1972 Dec 25;247(24):8031-5
references.pmid: 4344989
references.citation: PLoS Biol. 2003 Dec;1(3):E51
references.pmid: 14691533
references.citation: Biophys J. 1991 May;59(5):1091-100
references.pmid: 1651123
references.citation: Structure. 2005 Nov;13(11):1613-23
references.pmid: 16271884
references.citation: J Inorg Biochem. 1990 Mar;38(3):175-83
references.pmid: 2329344
references.citation: Biochem J. 2000 Sep 15;350 Pt 3:909-15
references.pmid: 10970808
references.citation: Biochim Biophys Acta. 1976 Nov 26;453(1):250-6
references.pmid: 999884
references.citation: Adv Inorg Biochem. 1983;5:201-33
references.pmid: 6382958
references.citation: J Biol Chem. 1978 Mar 25;253(6):1930-7
references.pmid: 204636
references.citation: Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258-62
references.pmid: 6300903
references.citation: Biochim Biophys Acta. 1983 Jul 14;762(4):498-502
references.pmid: 6307387
references.citation: J Biol Chem. 1975 Mar 25;250(6):2182-8
references.pmid: 803968
references.citation: Arch Biochem Biophys. 1990 Sep;281(2):251-6
references.pmid: 2168158
references.citation: J Biol Inorg Chem. 2009 Jun;14(5):771-81
references.pmid: 19290554
references.citation: Biochemistry. 2006 May 30;45(21):6663-73
references.pmid: 16716077
references.citation: Biopolymers. 2004 Feb 5;73(2):205-15
references.pmid: 14755578
references.citation: Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263-6
references.pmid: 6300904
references.citation: Biochem J. 1995 Jul 15;309 ( Pt 2):403-10
references.pmid: 7626003
references.citation: Int J Biochem Cell Biol. 2001 Oct;33(10):940-59
references.pmid: 11470229
references.citation: Biochim Biophys Acta. 1998 Apr 2;1383(2):197-210
references.pmid: 9602126
references.citation: Biochemistry. 1985 Dec 3;24(25):7412-8
references.pmid: 3853465
references.citation: Protein Expr Purif. 2001 Oct;23(1):142-50
references.pmid: 11570856
references.citation: J Mol Recognit. 2009 Nov-Dec;22(6):521-9
references.pmid: 19693784
references.citation: Biochemistry. 2007 Sep 18;46(37):10603-11
references.pmid: 17711300
references.citation: Biometals. 2007 Jun;20(3-4):249-62
references.pmid: 17216400
references.citation: Chem Rev. 1999 Sep 8;99(9):2817-42
references.pmid: 11749502
references.citation: Science. 2010 Feb 5;327(5966):653-4
references.pmid: 20133562
references.citation: Biochim Biophys Acta. 1999 Mar 19;1430(2):269-80
references.pmid: 10082955
references.citation: Biochim Biophys Acta. 1986 Apr 22;870(3):530-7
references.pmid: 3008845
references.citation: J Biol Chem. 2006 Aug 25;281(34):24934-44
references.pmid: 16793765
references.citation: Anal Biochem. 2008 Jul 15;378(2):202-7
references.pmid: 18471984
references.citation: Biochemistry. 1997 Sep 9;36(36):11036-43
references.pmid: 9283096
references.citation: Cell. 2004 Feb 20;116(4):565-76
references.pmid: 14980223
references.citation: Biochemistry. 1981 Nov 24;20(24):7033-9
references.pmid: 7317366
references.citation: Protein Expr Purif. 2004 Aug;36(2):318-26
references.pmid: 15249056
references.citation: Biochemistry. 1993 Apr 6;32(13):3387-95
references.pmid: 8461302
references.citation: Biochemistry. 1993 Aug 17;32(32):8162-7
references.pmid: 8347616
references.citation: J Mol Biol. 2010 Feb 12;396(1):130-40
references.pmid: 19917294
references.citation: Br J Haematol. 1977 May;36(1):85-96
references.pmid: 871428