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J Biol Chem, Vol. 273, Issue 27, 17018-17024, July 3, 1998

Mutations at Nonliganding Residues Tyr-85 and Glu-83 in the N-Lobe of Human Serum Transferrin
FUNCTIONAL SECOND SHELL EFFECTS

Qing-Yu He, Anne B. Mason, Robert C. Woodworth, Beatrice M. Tam^¶, Ross T. A. MacGillivray^¶, John K. Grady, and N. Dennis Chasteen

From the  Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont 05405, the ^¶ Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, and the  Department of Chemistry, Parsons Hall, University of New Hampshire, Durham, New Hampshire 03824

The x-ray crystal structure of the N-lobe of human serum transferrin has shown that there is a hydrogen bond network, the so-called "second shell," around the transferrin iron binding site. Tyrosine at position 85 and glutamic acid at position 83 are two nonliganding residues in this network in the human serum transferrin N-lobe (hTF/2N). Mutation of each of these two amino acids has a profound effect on the metal binding properties of hTF/2N. When Tyr-85 is mutated to phenylalanine, iron release from the resulting mutant Y85F is much more facile than from the parent protein. Elimination of the hydrogen bond between Tyr-85 and Lys-296 appears to interfere with the "di-lysine (Lys-206-Lys-296) trigger," which affects the iron binding stability of the protein. Surprisingly, mutation of Glu-83 to alanine leads to the absence of one of the normal iron binding ligands; introduction of a monovalent anion is able to restore the normal first coordination sphere. The missing ligand appears to be His-249, as revealed by comparison of the metal binding behaviors of mutants H249Q and E83A and structural analysis. Glu-83 has a strong H bond linkage with His-249 in apo-hTF/2N, which helps to hold the His-249 in the proper position for iron binding. Disabling Glu-83 by mutation to an alanine seriously disturbs the H bond network, allowing His-249 to move away. A monovalent anion can help reestablish the normal network by providing a negative charge near the position of Glu-83 to reach charge balance, so that ligand His-249 is available again for iron binding.

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