Structural study of the microglia/macrophage-specific protein Iba1 from human and mouse having an actin-binding activity

具有147个氨基酸残基的Iba1 （ionized calcium-binding adaptor molecule 1）是一种钙（Ca^<2+>）结合蛋白，在小胶质细胞/巨噬细胞中特异性表达，有望成为激活小胶质细胞的典型特征——膜褶皱的关键因素。我们已经确定了Ca^<2+>自由形式的人类Iba1晶体结构和Ca^<2+>结合形式的小鼠Iba1晶体结构，分别达到1.9 a和2.1 a的分辨率。Iba1的x射线结构显示了一个紧凑的单域蛋白，具有两个EF-hand基元，在整体拓扑结构上与经典的EF-hand蛋白肌钙蛋白C和钙调蛋白的部分结构相似。在小鼠Iba1中，第二个EF-hand含有结合的Ca^<2+>，但第一个EF-hand不含，这在S100蛋白中经常出现，这表明Iba1具有S100蛋白样的EF-hand。然而，Ca^<2+>-结合引起的Iba1分子构象变化与经典的EF-hand蛋白和/或S100蛋白不同，导致晶体中形成二聚体，这表明Iba1具有依赖于Ca^<2+>-结合的独特分子开关机制。二聚体具有略平的二次棱镜形状，具有特征电荷分布；负电荷区广泛分布在上表面和侧面，而不寻常的正电荷区位于下表面的中心。这个带正电的区域很可能与目标分子相互作用。为了阐明Iba1在溶液中的寡聚状态，进行了交联实验，表明Iba1在溶液中部分形成二聚体。为了研究二聚体Iba1是否可以与靶分子相互作用，我们也进行了表面等离子体共振研究，但尚未获得明确的证据。

Iba1 (ionized calcium-binding adaptor molecule 1) with 147 amino acid residues has been identified as a calcium (Ca^<2+>)-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in Ca^<2+> free form and mouse Iba1 in Ca^<2+> -bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca^<2+>, but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. However, the molecular conformational change induced by Ca^<2+>-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, leading to the formation of a dimer in a crystal, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca^<2+>-binding. The dimer has the shape of a slightly flat quadratic prism with the characteristic charge distribution; negatively charged regions are widely distributed on the upper surface and sides, while the unusual positively charged region is found at the center of the lower surface. It is likely that this positively charged region interacts with a target molecule. To elucidate the oligomeric state of Iba1 in a solution, cross-linking experiments was done, showing that Iba1 partially forms a dimer in a solution. To investigate whether a dimer Iba1 can interacts with target molecules or not, we also done surface plasmon resonance studies, but a clear evidence for this has not yet been obtained.