Pilipczuk Justyna, Zalewska-Piątek Beata, Bruździak Piotr, Czub Jacek, Wieczór Miłosz, Olszewski Marcin, Wanarska Marta, Nowicki Bogdan, Augustin-Nowacka Danuta, Piątek Rafał
From the Departments of Molecular Biotechnology and Microbiology and.
Physical Chemistry, Gdańsk University of Technology, 80-233 Gdańsk, Poland.
J Biol Chem. 2017 Sep 29;292(39):16136-16149. doi: 10.1074/jbc.M117.785477. Epub 2017 Jul 24.
Dr fimbriae are homopolymeric adhesive organelles of uropathogenic composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by ∼50 kJ mol in an exclusively thermodynamic manner, by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from ∼10 s to 10 s Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae.
Dr菌毛是由DraE亚基组成的泌尿道致病性同聚体黏附细胞器，负责与宿主细胞结合。这些结构的特点是由于DraE的Ig样折叠的结构特性而具有极高的稳定性。DraE和其他菌毛亚基在含Ig样结构域的蛋白质中与众不同的一个特征是连接其A链和B链的保守二硫键。在此，我们研究了该二硫键如何影响DraE的稳定性以及折叠/去折叠途径。我们发现，该二硫键以完全热力学的方式使自互补DraE（DraE-sc）稳定约50 kJ/mol，通过降低天然态的自由能且几乎不影响过渡态的自由能。通过实验测定的DraE-sc和缺乏二硫键的DraE-sc变体的折叠和去折叠速率常数证实了这一发现。尽管两种蛋白质的折叠表现出相似的动力学，但去除二硫键后去折叠速率常数变化了10个数量级，从约10⁻⁴ s⁻¹变为10⁻¹⁴ s⁻¹。分子模拟表明，缺乏二硫键的变体的去折叠由A链或G链引发，并且二硫键介导的A链与核心B链的连接协同稳定了整个蛋白质。我们还表明，DraE中的二硫键被DraB伴侣蛋白识别，这表明了一种机制，可防止将稳定性较低的非氧化形式的DraE纳入菌毛。
