Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry
Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry
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DOI：
10.1021/acs.biochem.5b00807
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发表时间：
2016-03-29
期刊：
影响因子：
2.9
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
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作者：
Fatunmbi, Ololade;Abzalimov, Rinat R.;Kaltashov, Igor A.
触珠蛋白 (Hp) 结合游离血红蛋白 (Hb) 二聚体，以防止细胞外环境中 Hb 循环的负面后果。尽管单体 Hb 和肌红蛋白 (Mb) 也存在潜在风险，但它们与 Hp 的相互作用尚未得到广泛研究。 Mb 与 Hb 的 α 链和 β 链同源，并共享许多保守的 Hb/Hp 界面残基，但 Hp 是否结合 Mb 仍不清楚。为了解决这个问题，使用计算生物学工具来预测 Hp 结合单体球蛋白所需的相互作用，并使用天然电喷雾电离质谱 (ESI-MS) 测试预测的关联。 Hb/Hp 晶体结构用作模板，创建与 Hp 异二聚体 (Mb(2)/Hp) 结合的两个 Mb 分子的分子模型。分子模型表明 Mb 可以结合在 Hp α 链结合位点，其中 73% 的珠蛋白/Hp 相互作用是保守的。相比之下，参与与 Hp 互补静电相互作用的几个离子 β 链残基对应于 Mb 中带有相反电荷的残基，表明在 β 链结合位点存在不利的静电 Hp/Mb 相互作用。如天然 ESI-MS 所示，在没有 Hb β 链的情况下，分离的单体 Hba 亚基可以与 Hp 异四聚体形成 2:1 复合物。天然 ESI-MS 还证实，在生理 pH 和离子强度下，Mb 可以与溶液中的 Hp 异四聚体结合，化学计量为 1:1 和 2:1。相对于 Hb α 链，Hp 对 Mb 的亲和力似乎有所减弱。我们的计算机实验合理化了这一变化，并证明蛋白质/蛋白质相互作用的分子建模对 MS 实验有重要帮助。
Haptoglobin (Hp) binds free hemoglobin (Hb) dimers to prevent negative consequences of Hb circulation in the extracellular environment. Although both monomeric Hb and myoglobin (Mb) species also present potential risks, their interactions with Hp have not been extensively studied. Mb is homologous to both the alpha- and beta-chains of Hb and shares many conserved Hb/Hp interface residues, yet whether Hp binds Mb remains unclear. To address this, computational biology tools were used to predict the interactions required for Hp to bind monomeric globins, and the predicted association was tested using native electrospray ionization mass spectrometry (ESI-MS). The Hb/Hp crystal structure was used as the template to create molecular models of two Mb molecules bound to an Hp heterodimer (Mb(2)/Hp). Molecular modeling suggests that Mb can bind at the Hp alpha-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic beta-chain residues involved in complementary electrostatic interactions with Hp correspond to residues with the opposite charge in Mb, suggesting unfavorable electrostatic Hp/Mb interactions at the beta-chain binding site. As shown by native ESI-MS, isolated monomeric Hba subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb beta-chains. Native ESI-MS also confirmed that Mb can bind to Hp heterotetramers in solution with stoichiometries of 1:1 and 2:1 at physiological pH and ionic strength. The affinity of Hp for Mb appears to be diminished relative to that of Hb alpha-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.