RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides
RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides
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DOI：
10.1074/jbc.m112.411769
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发表时间：
2013-01-25
影响因子：
4.8
通讯作者：
Zorreguieta, Angeles
中科院分区：
文献类型：
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作者：
Abdian, Patricia L.;Caramelo, Julio J.;Zorreguieta, Angeles
计算机模拟分析揭示了一个广泛存在于细菌中的保守蛋白结构域（CHDL），它与真核细胞钙粘蛋白具有显著的结构相似性。如我们和其他人所示，CHDL结构域被证明存在于RapA中，RapA是一种参与根瘤菌细胞自聚集、生物膜形成和粘附到植物根部的蛋白质。钙粘蛋白的结构相似性，建议钙依赖性的寡聚化CHDL域作为RapA行动的机制基础。在这里，我们表明圆二色谱，光散射，等温滴定量热法，和其他方法，RapA 2豆类根瘤菌确实表现出钙粘蛋白样β-折叠构象，其正确的折叠和稳定性依赖于每个蛋白质分子的一个钙离子的结合。通过进一步的计算机模拟分析，我们还揭示了RapA 2由两个CHDL结构域组成，并扩大了细菌和古细菌中含有CHDL的蛋白质的范围。然而，在不同浓度的添加的钙的光散射测定显示，RapA 2既不形成同源寡聚体，也不形成异源寡聚体与RapB（一种独特的CHDL蛋白），表明RapA 2不介导细胞相互作用通过钙粘蛋白样机制。相反，我们证明了RapA 2与由R.豆科植物中的钙依赖性的方式，维持这些蛋白质的作用，在发展的生物膜基质的EPS。由于RapA 2的EPS结合只能归因于其两个CHDL结构域，因此我们提出RapA 2是一种钙依赖性凝集素，并且各种细菌和古细菌蛋白质中的CHDL结构域赋予这些蛋白质碳水化合物结合活性。
In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like beta-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins.