Crystal Structure at 1.9 Å Resolution of the Apoovotransferrin N-Lobe Bound by Sulfate Anions: Implications for the Domain Opening and Iron Release Mechanism

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• 作者：Kimihiko Mizutani ; Honami Yamashita ; Bunzo Mikami ; and Masaaki Hirose
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：March 28, 2000
• 年：2000
• 卷：39
• 期：12
• 页码：3258 - 3265
• 全文大小：709K
• 年卷期：v.39,no.12(March 28, 2000)
• ISSN：1520-4995

文摘

Several lines of functional evidence have shown that anion binding to a nonsynergistic site isa prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structuralevidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 Å resolutionapo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to spacegroup P6₃22 with the following unit cell dimensions: a = b = 125.17 Å and c = 87.26 Å. The structuraldetermination was performed by isomorphous replacement, using Pt and Au derivatives. The structurerefinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 Å for the final model. From theelectron density map, the existence of four bound SO₄^2- anions was detected. Three of them that exhibitedreasonably low B-factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the boundanion directly interacts with an Fe³⁺-coordinating ligand; SO₄ O1 and SO₄ O3 form hydrogen bonds withHis250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in ahinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO₄ O2 andSO₄ O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchorgroups for CO₃^2- anion in holotransferrins. The former oxygen atom of SO₄^2- is also hydrogen bondedto Ser122 N, which forms a hydrogen bond with Fe³⁺-coordinating ligand Asp60 OD2 in holotransferrins.Some of the SO₄^2- oxygen atoms in sites 1 and 2 interact indirectly through H₂O molecules with functionallyimportant protein groups, such as the other Fe³⁺-coordinating ligands, Tyr92 OH and Tyr191 OH, and adilysine trigger group, Lys209 NZ. In site 3, SO₄ O1 and SO₄ O4 form hydrogen bonds with Ser192 OGand Tyr191 N, respectively, and SO₄ O2 forms hydrogen bonds with Ser192 N and Ser192 OG. Thesestructural data are consistent with the view that the anion bindings to the interdomain cleft, especially tosites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the ironrelease mechanism of the ovotransferrin N-lobe.