Harris Gemma, Ma Wenjiang, Maurer Lisa M, Potts Jennifer R, Mosher Deane F
From the Department of Biology, University of York, York YO10 5DD, United Kingdom and.
the Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, Wisconsin 53706.
J Biol Chem. 2014 Aug 8;289(32):22490-9. doi: 10.1074/jbc.M114.578419. Epub 2014 Jun 24.
BBK32 is a fibronectin (FN)-binding protein expressed on the cell surface of Borrelia burgdorferi, the causative agent of Lyme disease. There is conflicting information about where and how BBK32 interacts with FN. We have characterized interactions of a recombinant 86-mer polypeptide, "Bbk32," comprising the unstructured FN-binding region of BBK32. Competitive enzyme-linked assays utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies showed that Bbk32 binding involves both the fibrin-binding and the gelatin-binding domains of the 70-kDa N-terminal region (FN70K). Crystallographic and NMR analyses of smaller Bbk32 peptides complexed, respectively, with (2-3)FNI and (8-9)FNI, demonstrated that binding occurs by β-strand addition. Isothermal titration calorimetry indicated that Bbk32 binds to isolated FN70K more tightly than to intact FN. In a competitive enzyme-linked binding assay, complex formation with Bbk32 enhanced binding of FN with mAbIII-10 to the (10)FNIII module. Thus, Bbk32 binds to multiple FN type 1 modules of the FN70K region by a tandem β-zipper mechanism, and in doing so increases accessibility of FNIII modules that interact with other ligands. The similarity in the FN-binding mechanism of BBK32 and previously studied streptococcal proteins suggests that the binding and associated conformational change of FN play a role in infection.
BBK32是一种在莱姆病病原体伯氏疏螺旋体细胞表面表达的纤连蛋白(FN)结合蛋白。关于BBK32在何处以及如何与FN相互作用,存在相互矛盾的信息。我们已经对一种重组的86聚体多肽“Bbk32”的相互作用进行了表征,该多肽包含BBK32的无结构FN结合区域。利用各种FN片段和表位映射的抗FN单克隆抗体进行的竞争性酶联测定表明,Bbk32结合涉及70 kDa N端区域(FN70K)的纤维蛋白结合域和明胶结合域。分别与(2-3)FNI和(8-9)FNI复合的较小Bbk32肽的晶体学和核磁共振分析表明,结合是通过β链添加发生的。等温滴定量热法表明,Bbk32与分离的FN70K的结合比与完整FN的结合更紧密。在竞争性酶联结合测定中,与Bbk32形成复合物增强了FN与单克隆抗体III-10与(10)FNIII模块的结合。因此,Bbk32通过串联β拉链机制与FN70K区域的多个FN 1型模块结合,并且这样做增加了与其他配体相互作用的FNIII模块的可及性。BBK32与先前研究的链球菌蛋白在FN结合机制上的相似性表明,FN的结合和相关的构象变化在感染中起作用。