He Q Y, Mason A B, Tam B M, MacGillivray R T, Woodworth R C
Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405, USA.
Biochemistry. 1999 Jul 27;38(30):9704-11. doi: 10.1021/bi990134t.
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.
转铁蛋白N端叶（hTF/2N）中二赖氨酸（Lys206-Lys296）对的独特结构特征被推测在铁从该蛋白的释放过程中发挥特殊作用。这两个赖氨酸位于相对的结构域中，在中性pH条件下，含铁血的hTF/2N中它们相互形成氢键，但在该蛋白的脱辅基形式中则相距甚远。有人提出，在较低pH下二赖氨酸质子化导致的电荷排斥可能是打开裂隙并促进铁释放的触发因素。二赖氨酸对带正电荷且位于靠近金属结合中心的位置这一事实，也使人认为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来证实二赖氨酸对在调节铁释放中发挥这一双重作用。当其中任何一个赖氨酸突变为谷氨酸或谷氨酰胺，或者两个都突变为谷氨酸时，与野生型蛋白相比，铁的释放要慢得多。这是因为在突变体中不存在打开裂隙的驱动力，或者这种驱动力转变为一种类似锁定的相互作用（在K206E和K296E突变体的情况下）。用阴离子对脱辅基蛋白进行直接滴定以及依赖阴离子的铁释放研究表明，二赖氨酸对是一个活性阴离子结合位点的一部分，该位点以Lys296-Tyr188相互作用为核心存在。在这个位点，Lys296作为主要的阴离子结合残基，Tyr188是电子光谱变化的主要报告基团，Lys206、Tyr85和Tyr95的贡献较小。在载铁的hTF/2N中，通过紫外可见差光谱监测发现阴离子结合变得不可见，因为光谱报告基团Tyr188和Tyr95与铁结合。我们的数据有力地支持了脱辅基hTF/2N在开放构象和封闭构象之间存在平衡的假设，因为只有在封闭形式中Lys296才与Tyr188直接接触。当前的研究结果整合了大量先前研究的观察结果、观点和实验数据，并进一步阐明了转铁蛋白N端叶中铁释放的详细机制。在含铁血的hTF/2N中，Lys296可能仍然作为一个靶点，在铁结合中心附近引入一个阴离子（或螯合剂）。当pH降低时，碳酸根（金属结合的协同阴离子）的质子化，然后是二赖氨酸对形成打开裂隙的驱动力，使铁暴露出来；附近的阴离子（或螯合剂）随后与铁结合并将其从蛋白中释放出来。
