在人红细胞SLC4A1和蛋白激酶SYK和LYN的整合膜蛋白中寻找可能的血红素结合位点

36 Pub Date : 2021-08-25 DOI:10.26565/2075-5457-2021-36-1
T. Barannik, M. Lebedieva
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引用次数: 0

摘要

SLC4A1蛋白或3带蛋白(band 3)是最常见的红细胞膜蛋白之一。与阴离子交换剂的功能一起,它通过与细胞骨架成分和酶形成各种复合物,有助于控制细胞形状和寿命。在氧化应激下,蛋白质发生氧化修饰,特别是由于血红蛋白聚集体的结合,但作为血红蛋白降解的主要产物血红素对带3蛋白活性的直接影响尚未见文献报道。已知溶血条件伴随着SLC4A1蛋白、非受体酪氨酸蛋白激酶LYN(残基Y359)和SYK(残基Y8和Y21)的磷酸化,而SYK激酶抑制剂对红细胞具有稳定作用。血红素对Src激酶(包括SYK和LIN激酶)的调控作用已被阐明,但它们与血红素相互作用的位点尚未被研究。因此,本研究的目的是预测红细胞膜SLC4A1整合蛋白中可能的血红素结合位点,以及SYK和LYN蛋白激酶及其与SLC4A1的复合物中可能的血红素结合位点。对SLC4A1蛋白的序列分析显示,SLC4A1蛋白中有几个非肽具有潜在的血红素结合位点,包括胞质His98和TM5和TM6跨膜段之间的Tyr553和Tyr555残基。这些残基以及氨基酸Tyr216、His303和His192也被HemeBind工具预测为血红素结合位点。SYK蛋白激酶的血红素结合位点最多,包括两个胞质残基Tyr216和His303。SLC4A1蛋白的分子对接显示,在细胞质结构域His192和173-176区(PDB ID 4KY9和1HYN)之间的空腔中有一个血红素结合位点,也是SYK或LYN激酶模拟复合物的一部分。值得注意的是,175-185位点被称为锚蛋白结合位点。将血红素与膜结构域(PDB ID 4YZF)对接,发现TM5中Lys539附近有一个推测的血红素结合位点,根据文献,该位点属于对阴离子运输抑制剂DIDS敏感的反应中心之一。在活性位点(PDB ID 4FL2)与SYK蛋白激酶的ATP分子对接,揭示了两个推测的血红素结合位点——Tyr64附近和His243附近,但当从PDB文件中去除ATP后,血红素占据了Lys402和His531附近的一个核苷酸结合位点。LYN蛋白激酶(PDB ID 5XY1)与抑制剂分子(哌嗪衍生物)的结构在Tyr321附近有一个血红素结合位点。去除抑制剂后,血红素占据了Glu290和Ala371附近的区域。在大多数模型复合物中,band 3蛋白比LYN和SYK蛋白激酶更可能是血红素结合位点,但具有游离活性位点的激酶显然可以结合血红素而不是底物,这将干扰磷酸化。血红素积累过程中3带蛋白的破坏可以抑制阴离子运输或使SLC4A1复合物与细胞骨架蛋白的形成复杂化,这与磷酸化的影响一起可能是降低红细胞稳定性的机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Search for putative heme binding sites in the integrated membrane protein of human erythrocytes SLC4A1 and protein kinases SYK and LYN
SLC4A1 protein or band 3 protein (band 3) is one of the most common erythrocyte membrane proteins. Along with the function of the anion exchanger, it contributes to the control of cell shape and lifespan through the formation of various complexes with cytoskeletal components and enzymes. Under oxidative stress, the protein oxidative modifications occur, in particular, due to the binding of hemoglobin aggregates, but the direct effect of heme as the major product of hemoglobin degradation on the band 3 protein activity has not been described in the literature. It is known that hemolytic conditions are accompanied by phosphorylation of the SLC4A1 protein, non-receptor tyrosine protein kinases LYN (by residue Y359), and SYK (by residues Y8 and Y21), while SYK kinase inhibitors have a stabilizing effect on erythrocytes. The regulatory effect of heme on Src kinases, which include SYK and LIN kinases, has been clarified, but the sites of their interaction with heme have not been investigated. Therefore, the aim of this study was to predict putative heme binding sites in the integral protein of the erythrocyte membrane SLC4A1, as well as in the protein kinases SYK and LYN and their complexes with SLC4A1, modeled in silico. Sequence analysis of proteins with HeMoQuest revealed several nonapeptides with potential heme binding sites in the SLC4A1 protein, including the cytosolic His98 and Tyr553 and Tyr555 residues in the region between the TM5 and TM6 transmembrane segments. These residues, as well as the amino acids Tyr216, His303, and His192, were also predicted as heme-binding sites by HemeBind tool. The largest number of putative heme binding sites was found for SYK protein kinase, including the two cytosolic residues Tyr216 and His303. Molecular docking of the SLC4A1 protein revealed a heme binding site in the cavity between His192 and region 173–176 in the cytosolic domain structure (PDB ID 4KY9 and 1HYN), also as part of a simulated complex with SYK or LYN kinases. It should be noted that site 175–185 is known as the ankyrin binding site. Docking heme to the membrane domain (PDB ID 4YZF) revealed a putative heme binding site near Lys539 in TM5, which, according to the literature, belongs to one of the reactive centers sensitive to the anionic transport inhibitor DIDS. Molecular docking to SYK protein kinase with ATP in the active site (PDB ID 4FL2) revealed two putative heme binding sites – near Tyr64 and near His243, but when ATP was removed from pdb-file, the heme occupied a nucleotide binding site in the cavity near Lys402 and His531. LYN protein kinase (PDB ID 5XY1) had a heme binding site near Tyr321 in the structure with inhibitor molecule (piperazine derivative). After removing the inhibitor, the heme occupied its area near Glu290 and Ala371. In most model complexes, the band 3 protein was revealed to be a more likely heme binding site than LYN and SYK protein kinases, but kinases with free active sites can apparently bind a heme instead of substrate, which will interfere phosphorylation. Disruption of band 3 protein under heme accumulation can inhibit an anion transport or complicate the formation of SLC4A1 complexes with cytoskeletal proteins that together with the effects on phosphorylation could be a mechanism for reducing erythrocyte stability.
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