Fatunmbi Ololade, Abzalimov Rinat R, Savinov Sergey N, Gershenson Anne, Kaltashov Igor A
Department of Chemistry and ‡Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.
Biochemistry. 2016 Mar 29;55(12):1918-28. doi: 10.1021/acs.biochem.5b00807. Epub 2016 Mar 17.
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 α- and β-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 (Mb2/Hp). Molecular modeling suggests that Mb can bind at the Hp α-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic β-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 β-chain binding site. As shown by native ESI-MS, isolated monomeric Hbα subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb β-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 α-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.
触珠蛋白（Hp）可结合游离血红蛋白（Hb）二聚体，以防止Hb在细胞外环境中循环产生负面影响。尽管单体Hb和肌红蛋白（Mb）也都存在潜在风险，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链均同源，且共享许多保守的Hb/Hp界面残基，但Hp是否能结合Mb仍不清楚。为了解决这个问题，我们使用计算生物学工具预测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）对预测的结合进行测试。以Hb/Hp晶体结构为模板，构建了与Hp异二聚体结合的两个Mb分子的分子模型（Mb2/Hp）。分子建模表明，Mb可结合于Hp的α链结合位点，其中73%的球蛋白/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β链离子残基在Mb中对应电荷相反的残基，这表明在β链结合位点存在不利的Hp/Mb静电相互作用。如天然ESI-MS所示，在没有Hbβ链的情况下，分离的单体Hbα亚基可与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可与溶液中的Hp异四聚体以1:1和2:1的化学计量比结合。相对于Hbα链，Hp对Mb的亲和力似乎有所降低。我们的计算机模拟实验解释了这种变化，并证明蛋白质/蛋白质相互作用的分子建模对质谱实验是一种有价值的辅助手段。
