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首页> 外文期刊>Biochemistry >Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.
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Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

机译：Lys206-Lys296相互作用在人转铁蛋白N瓣中的双重作用：铁释放触发和阴离子结合位点。

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摘要

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.
机译：假定转铁蛋白N瓣（hTF / 2N）中的二赖氨酸（Lys206-Lys296）对的独特结构特征在从蛋白质释放铁方面起特殊作用。这两个赖氨酸位于相反的区域，在中性pH值下，含铁的hTF / 2N中的氢彼此键合，但在蛋白质的脱辅基形式中相距甚远。已经提出，在较低pH下由二赖氨酸的质子化引起的电荷排斥可能是打开裂缝并促进铁释放的触发。二赖氨酸对带正电并且位于靠近金属结合中心的位置，这一事实也导致人们提出，二赖氨酸对是螯合剂的阴离子结合位点。本报告提供了全面的证据，以确认二赖氨酸对在调节铁释放中起双重作用。当任一赖氨酸突变为谷氨酸或谷氨酰胺时，或当两者都突变为谷氨酸时，铁的释放都比野生型蛋白慢得多。这是由于以下事实：在突变体中不存在用于裂口打开的驱动力，或者转换为锁状相互作用（对于K206E和K296E突变体而言）。用阴离子直接滴定脱辅基蛋白以及依赖阴离子的铁释放研究表明，二赖氨酸对是一个以Lys296-Tyr188相互作用为核心存在的活性阴离子结合位点的一部分。在此站点上，Lys296是主要的阴离子结合残基，而Tyr188是电子光谱变化的主要报告者，而Lys206，Tyr85和Tyr95的贡献较小。在载铁的hTF / 2N中，由于光谱报告子Tyr188和Tyr95与铁结合，因此通过UV-vis差光谱监测，阴离子结合变得不可见。我们的数据强有力地支持了apo-hTF / 2N在开放和封闭构象之间处于平衡状态的假说，因为只有在封闭形式下Lys296与Tyr188直接接触。目前的发现汇集了来自大量先前研究的观察结果，观点和实验数据，进一步揭示了铁从转铁蛋白N瓣释放的详细机理。在含铁的hTF / 2N中，Lys296仍可充当将阴离子（或螯合剂）引入铁结合中心附近的靶标。当pH降低时，碳酸盐的质子化（用于金属键合的协同阴离子），然后二赖氨酸对形成驱动力，使裂口松动，暴露出铁。然后附近的阴离子（或螯合剂）与铁结合并从蛋白质中释放出来。

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