Korotkova Natalia, Le Trong Isolde, Samudrala Ram, Korotkov Konstantin, Van Loy Cristina P, Bui Anh-Linh, Moseley Steve L, Stenkamp Ronald E
Department of Microbiology, University of Washington, Seattle, Washington 98195.
Department of Biological Structure, University of Washington, Seattle, Washington 98195; Biomolecular Structure Center, University of Washington, Seattle, Washington 98195.
J Biol Chem. 2006 Aug 4;281(31):22367-22377. doi: 10.1074/jbc.M604646200. Epub 2006 Jun 2.
DaaE is a member of the Dr adhesin family of Escherichia coli, members of which are associated with diarrhea and urinary tract infections. A receptor for Dr adhesins is the cell surface protein, decay-accelerating factor (DAF). We have carried out a functional analysis of Dr adhesins, as well as mutagenesis and crystallographic studies of DaaE, to obtain detailed molecular information about interactions of Dr adhesins with their receptors. The crystal structure of DaaE has been solved at 1.48 A resolution. Trimers of the protein are found in the crystal, as has been the case for other Dr adhesins. Naturally occurring variants and directed mutations in DaaE have been generated and analyzed for their ability to bind DAF. Mapping of the mutation sites onto the DaaE molecular structure shows that several of them contribute to a contiguous surface that is likely the primary DAF-binding site. The DAF-binding properties of purified fimbriae and adhesin proteins from mutants and variants correlated with the ability of bacteria expressing these proteins to bind to human epithelial cells in culture. DaaE, DraE, AfaE-III, and AfaE-V interact with complement control protein (CCP) domains 2-4 of DAF, and analysis of the ionic strength dependence of their binding indicates that the intermolecular interactions are highly electrostatic in nature. The adhesins AfaE-I and NfaE-2 bind to CCP-3 and CCP-4 of DAF, and electrostatic interactions contribute significantly less to these interactions. These observations are consistent with structural predictions for these Dr variants and also suggest a role for the positively charged region linking CCP-2 and CCP-3 of DAF in electrostatic Dr adhesin-DAF interactions.
DaaE是大肠杆菌Dr黏附素家族的成员，该家族成员与腹泻和尿路感染有关。Dr黏附素的一种受体是细胞表面蛋白衰变加速因子（DAF）。我们对Dr黏附素进行了功能分析，并对DaaE进行了诱变和晶体学研究，以获取有关Dr黏附素与其受体相互作用的详细分子信息。DaaE的晶体结构已在1.48埃分辨率下解析。在晶体中发现该蛋白以三聚体形式存在，其他Dr黏附素也是如此。已产生并分析了DaaE中的天然变体和定向突变体与DAF结合的能力。将突变位点映射到DaaE分子结构上表明，其中几个位点形成了一个连续的表面，这可能是主要的DAF结合位点。来自突变体和变体的纯化菌毛和黏附素蛋白的DAF结合特性与表达这些蛋白的细菌在培养物中与人上皮细胞结合的能力相关。DaaE、DraE、AfaE-III和AfaE-V与DAF的补体控制蛋白（CCP）结构域2-4相互作用，对其结合的离子强度依赖性分析表明，分子间相互作用本质上是高度静电性的。黏附素AfaE-I和NfaE-2与DAF的CCP-3和CCP-4结合，静电相互作用对这些相互作用的贡献明显较小。这些观察结果与这些Dr变体的结构预测一致，也表明DAF中连接CCP-2和CCP-3的带正电区域在静电Dr黏附素-DAF相互作用中起作用。
