Baker Tessa M, Nakashige Toshiki G, Nolan Elizabeth M, Neidig Michael L
Department of Chemistry , University of Rochester , Rochester , New York 14627 , USA . Email:
Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , USA . Email:
Chem Sci. 2017 Feb 1;8(2):1369-1377. doi: 10.1039/c6sc03487j. Epub 2016 Oct 11.
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 HisAsp motif (site 1) and a His 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 (10 = 11 045 cm) for a non-heme Fe(ii) protein. Analysis of CP variants lacking residues of the His 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 His motif, the metal ion also binds at site 1 of CP to form a five-coordinate (5C) Fe(ii)-HisAsp motif that was previously unidentified in this system. Notably, the introduction of His-to-Ala mutations at the His 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)-His site for metalloproteins and support the notion that Ca(ii) ions influence the Fe(ii)-binding properties of CP.
钙卫蛋白（CP）是一种参与宿主防御的丰富的金属螯合蛋白，最近发现人CP具有以钙依赖方式结合亚铁离子（Fe(ii)）的能力。在本研究中，采用近红外磁圆二色光谱研究CP两个过渡金属结合位点（一个HisAsp基序（位点1）和一个His基序（位点2））上Fe(ii)配位的性质。在加入亚化学计量的Fe(ii)时，在过量钙离子（Ca(ii)）存在下，优先形成与位点2相关的六配位（6C）Fe(ii)中心。对于非血红素Fe(ii)蛋白，该位点表现出异常大的配体场（10 = 11045 cm）。对缺乏His基序残基的CP变体的分析支持CP通过使用六个His配体在位点2配位Fe(ii)。在存在大于一个当量的Fe(ii)时或His基序发生突变时，金属离子也会结合到CP的位点1，形成一个五配位（5C）的Fe(ii)-HisAsp基序，该基序在此系统中以前未被鉴定。值得注意的是，在His基序处引入His到Ala突变会在亚化学计量的Fe(ii)存在下导致6C（位点2）和5C（位点1）信号的混合。这些结果与位点2的Fe(ii)结合亲和力降低一致，因为较弱配位的水衍生配体完成了6C位点。在没有Ca(ii)的情况下，加入亚化学计量的Fe(ii)时位点1和2都会被占据，并且观察到5C位点有更强的配体场。这些光谱研究进一步评估了金属蛋白独特的非血红素Fe(ii)-His位点，并支持钙离子影响CP的Fe(ii)结合特性这一观点。
