Magnetic circular dichroism studies of iron(ii) binding to human calprotectin.
Magnetic circular dichroism studies of iron(ii) binding to human calprotectin.
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DOI：
10.1039/c6sc03487j
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发表时间：
2017-02-01
期刊：
影响因子：
8.4
通讯作者：
Neidig ML
中科院分区：
文献类型：
--
作者：
Baker TM;Nakashige TG;Nolan EM;Neidig ML
铁（ii）与人体钙保护蛋白结合的磁圆二色性研究证明了铁（ii）在两个不同的铁（ii）结合位点的配位性质，并提供了钙（ii）如何调节铁（ii）配位的见解。钙保护蛋白（Calprotectin， CP）是一种丰富的参与宿主防御的金属螯合蛋白，最近发现人类钙保护蛋白以钙依赖的方式结合Fe（ii）的能力。在本研究中，采用近红外磁圆二色光谱研究了CP的两个过渡金属结合位点His3Asp基序（位点1）和His6基序（位点2）上Fe（ii）配位的性质。在添加亚化学计量量的Fe（ii）后，在过量Ca（ii）存在的情况下，优先形成与位点2相关的六坐标（6C） Fe（ii）中心。对于非血红素铁（ii）蛋白，该位点具有非常大的配体场（10D q = 11045 cm-1）。对缺乏His6基序残基的CP变异体的分析支持CP通过使用6个His配体在位点2协调Fe（ii）。当存在一个以上的Fe（ii）基序或His6基序突变时，金属离子也会结合在CP的1号位点形成一个五坐标（5C） Fe(ii) -His3Asp基序，该基序先前在该系统中未被发现。值得注意的是，在His6基序上引入His-to-Ala突变导致在亚化学计量量Fe存在下6C（位点2）和5C（位点1）信号的混合（ii）。这些结果与位点2的铁（ii）结合亲和力降低一致，因为更弱配位的水衍生配体完成6C位点。在没有Ca（ii）的情况下，当添加亚化学计量量的Fe（ii）时，位点1和2都被占据，并且在5C位点观察到更强的配体场。这些光谱研究为金属蛋白独特的非血红素Fe(ii) -His6位点提供了进一步的评估，并支持了Ca（ii）离子影响CP的Fe（ii）结合特性的观点。
Magnetic circular dichroism studies of Fe(ii) binding to human calprotectin demonstrate the nature of Fe(ii) coordination at two different Fe(ii)-binding sites and provide insight into how Ca(ii) modulates Fe(ii) coordination. Calprotectin (CP) is an abundant metal-chelating protein involved in host defense, and the ability of human CP to bind Fe(ii) in a calcium-dependent manner was recently discovered. In the present study, near-infrared magnetic circular dichroism spectroscopy is employed to investigate the nature of Fe(ii) coordination at the two transition-metal-binding sites of CP that are a His3Asp motif (site 1) and a His6 motif (site 2). Upon the addition of sub-stoichiometric Fe(ii), a six-coordinate (6C) Fe(ii) center associated with site 2 is preferentially formed in the presence of excess Ca(ii). This site exhibits an exceptionally large ligand field (10D q = 11 045 cm–1) for a non-heme Fe(ii) protein. Analysis of CP variants lacking residues of the His6 motif supports that CP coordinates Fe(ii) at site 2 by employing six His ligands. In the presence of greater than one equiv. of Fe(ii) or upon mutation of the His6 motif, the metal ion also binds at site 1 of CP to form a five-coordinate (5C) Fe(ii)–His3Asp motif that was previously unidentified in this system. Notably, the introduction of His-to-Ala mutations at the His6 motif results in a mixture of 6C (site 2) and 5C (site 1) signals in the presence of sub-stoichiometric Fe(ii). These results are consistent with a reduced Fe(ii)-binding affinity of site 2 as more weakly coordinating water-derived ligands complete the 6C site. In the absence of Ca(ii), both sites 1 and 2 are occupied upon addition of sub-stoichiometric Fe(ii), and a stronger ligand field is observed for the 5C site. These spectroscopic studies provide further evaluation of a unique non-heme Fe(ii)–His6 site for metalloproteins and support the notion that Ca(ii) ions influence the Fe(ii)-binding properties of CP.