Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
J Biol Chem. 2011 Jun 17;286(24):21353-60. doi: 10.1074/jbc.M111.226449. Epub 2011 Apr 12.
Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.
来自巴斯德氏菌科的革兰氏阴性猪病原体具有一种表面受体复合物，能够从猪转铁蛋白（pTf）中获取铁。该受体由转铁蛋白结合蛋白 A（TbpA）、一种跨膜铁转运蛋白和 TbpB、一种表面暴露的脂蛋白组成。目前仍不清楚该受体复合物如何以能够释放铁的方式与 pTf 结合，以及不同的菌株是否在 Tf 上具有不同的识别位点。在这项研究中，使用质量位移分析和分子对接模拟相结合的方法绘制了 TbpB-pTf 界面图，确定了多种不同的副猪嗜血杆菌和猪链球菌菌株的结合部位仅位于 pTf 的 C 结构域。进一步通过定点突变进行了界面特征分析和验证。尽管靶向同一结构域，但不同变体对构成铁配位位点的两个亚结构域具有不同的偏好。亚结构域 C1 和 C2 参与高亲和力结合，但 C1 亚结构域对整体亲和力的贡献较小。此外，根据竞争性铁结合研究，TbpB-pTf 复合物不能独立于其他介质释放铁。综上所述，我们的研究结果支持这样一种模型，即 TbpB 能够高效地捕获和呈现负载铁的 pTf 给摄取途径的其他成分，即使在低铁条件下也是如此。
