Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine 'trigger' but does not significantly affect iron release

Ross T.A. MacGillivray, Maria Bewley, Clyde A. Smith, Qing Yu He, Anne B. Mason, Robert C. Woodworth, Edward N. Baker

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive 'trigger' involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half- molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.

Original languageEnglish (US)
Pages (from-to)1211-1216
Number of pages6
JournalBiochemistry
Volume39
Issue number6
DOIs
StatePublished - Feb 15 2000

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lysyllysine
Transferrin
Iron
Ligands
Mutation
R388
Crystal structure
Acids
Protonation
Mutant Proteins
Histidine
Lysine
Hydrogen
Carrier Proteins
Proteins
Binding Sites

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

MacGillivray, Ross T.A. ; Bewley, Maria ; Smith, Clyde A. ; He, Qing Yu ; Mason, Anne B. ; Woodworth, Robert C. ; Baker, Edward N. / Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine 'trigger' but does not significantly affect iron release. In: Biochemistry. 2000 ; Vol. 39, No. 6. pp. 1211-1216.
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abstract = "Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive 'trigger' involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half- molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8{\%}, R(free) 29.4{\%}), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.",
author = "MacGillivray, {Ross T.A.} and Maria Bewley and Smith, {Clyde A.} and He, {Qing Yu} and Mason, {Anne B.} and Woodworth, {Robert C.} and Baker, {Edward N.}",
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Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine 'trigger' but does not significantly affect iron release. / MacGillivray, Ross T.A.; Bewley, Maria; Smith, Clyde A.; He, Qing Yu; Mason, Anne B.; Woodworth, Robert C.; Baker, Edward N.

In: Biochemistry, Vol. 39, No. 6, 15.02.2000, p. 1211-1216.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine 'trigger' but does not significantly affect iron release

AU - MacGillivray, Ross T.A.

AU - Bewley, Maria

AU - Smith, Clyde A.

AU - He, Qing Yu

AU - Mason, Anne B.

AU - Woodworth, Robert C.

AU - Baker, Edward N.

PY - 2000/2/15

Y1 - 2000/2/15

N2 - Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive 'trigger' involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half- molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.

AB - Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive 'trigger' involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half- molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.

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