structural characterization of iron uptake from human transferrin

2011年完成了以下工作： 细菌和人蛋白质的表达、纯化及复合物的形成 我们在2007年解决了全长apo人转铁蛋白的结构并公开了该结构（Wally et al.，2007）。在过去的一年里，我们使用这些信息对下面描述的TbpA-hTf的复杂结构进行了定相，同时我们解决了载脂蛋白hTf C端的高得多的分辨率结构，分辨率为1.7。 我们开发了一个表达系统，能够产生0.2-0.4 mg/L的纯化蛋白的TbpA。 本文报道了E.使用含有TbpA基因以及N-末端His-标签的质粒转化的大肠杆菌生长，用低水平IPTG诱导过夜，然后收获。 我们最近通过C-末端β链的定向诱变提高了表达产量，使新合成的蛋白质能够更有效地被E.大肠杆菌膜插入器。 我们开发了TbpB的表达系统，其能够产生7-8 mg/L的纯化蛋白。 采用中规模（4L）制备E.生长用含有TbpB基因（缺乏N-末端膜锚的可溶性构建体）以及N-末端His-标签和切割位点的质粒转化的大肠杆菌，并用高水平IPTG诱导数小时，然后收获。 然后裂解细胞并通过超离心分离膜，并使可溶性级分在镍-NTA柱上流动。 然后进一步纯化镍螯合物，并可与纯化的市售铁结合人血清转铁蛋白结合。今年，我们将TbpB的结构解析到了2.65分辨率。 使用我们纯化的TbpB，其仅结合holo hTf，我们制备了TbpB-holo hTf复合物，并使用小角X射线散射对其进行表征。由于我们有两种蛋白质组分的结构，我们能够在结构上表征复合物，即使我们还不能使其结晶。 我们最近获得了TbpA+hTf的晶体（总共180 kDa），其将X射线衍射到2.65分辨率。在2010年8月通过使用我们的hTf坐标的分子置换来解析结构（Wally et al.，JBC 2007）和各种TonB依赖性转运蛋白模型。这种结构是第一个与全长宿主靶标复合的外膜蛋白（举例说明宿主病原体相互作用），它告诉我们有关人转铁蛋白的特异性，参与这种相互作用的残基，可变和保守表位位于细胞外表面（用于疫苗设计），铁如何从hTf中提取，以及它如何通过外膜转运。当TbpA结合hTf时，它通过由81个TbpA残基和67个hTf残基组成的广泛界面螯合2500 2的掩埋表面。这座建筑是一项里程碑式的成就，花了12年时间才完成。 最近，我们首次在体外制备了TbpA-TbpB与全人转铁蛋白（hTf）结合的复合物，并通过负染单粒子电镜对其进行了表征。从TbpA-hTf晶体结构和TbpB-hTf SAXS结构，我们制作了三重复合物的模型，该模型非常适合EM 3D重建。从所有这些数据中，我们现在了解了识别的特异性，并且已经设计出可以阻断TbpA-转铁蛋白相互作用的抗体。由于TbpA和TbpB存在于所有临床分离株中，因此它们代表重要的治疗靶点。这是最大的外膜蛋白复合物之一，其特征在于在高分辨率和它有直接的翻译应用。一份描述晶体学、SAXS、EM和功能分析的手稿最近提交给了《自然》杂志。 2012年更新：这项工作于2012年2月在《自然》杂志上发表。我们现在已经提供了一个完整的结构描述的铁输入途径奈瑟菌，并建立了几个可验证的假设铁提取和输入。这项工作已经被认为是一个具有里程碑意义的论文，对该领域产生了重大影响，并改变了奈瑟氏菌未来实验的进程。

The following work was accomplished in 2011: Expression, Purification and Complex Formation of Bacterial and Human Proteins We solved the structure of full-length, apo human transferrin in 2007 and published this structure (Wally et al., J. Biol. Chem. 2007). Thispast year we used the information to phase a complex structure for TbpA-hTf described below, and at the same time we solved a much higher resolution structure of apo hTf C-lobe to 1.7 resolution. We developed an expression system for TbpA that is capable of producing 0.2-0.4 mg/L of purified protein. A large scale preparation (27L) of E. coli transformed with a plasmid containing the gene for TbpA as well as an N-terminal His-Tag is grown and induced with low level IPTG overnight and then harvested. We recently improved expression yields through targeted mutagenesis of the C-terminal beta strand, allowing the newly synthesized protein to be more efficiently processed by the E. coli membrane insertion machinery. We developed expression system for TbpB that is capable of producing 7-8 mg/L of purified protein. A medium scale preparation (4L) of E. coli transformed with a plasmid containing the gene for TbpB (a soluble construct lacking the N-terminal membrane anchor) as well as an N-terminal His-Tag and a cleavage site are grown and induced with high level IPTG for a few hours and then harvested. The cells are then lysed and membranes are separated via ultracentrifugation, and the soluble fraction is run over a Nickel-NTA column. The nickel eluate is then further purified and can be bound to purified commercially available iron-bound human serum transferrin. We solved the structure of TbpB to 2.65 resolution this year. Using our purified TbpB, which only binds holo hTf, we made a TbpB-holo hTf complex and characterized it using small angle X-ray scattering. Since we have structures of the two protein components, we were able to structurally characterize the complex, even though we have not been able to crystallize it. We recently obtained crystals of TbpA+hTf (180 kDa total) that diffract X-rays to 2.65 resolution. The structure was solved in August 2010 by molecular replacement using our hTf coordinates (Wally et al., JBC 2007) and various TonB-dependent transporter models. This structure, the first of an outer membrane protein in complex with its full-length host target (exemplifying host pathogen interactions), tells us about the specificity for human transferrin, the residues involved in this interaction, where variable and conserved epitopes are located on the extracellular surface (useful for vaccine design), how iron is extracted from hTf, and how it is transported across the outer membrane. When TbpA binds hTf, it sequesters 2500 2 of buried surface through an extensive interface consisting of 81 TbpA residues and 67 hTf residues. This structure is a landmark achievement, taking 12 years to complete. Recently, we made the first ever in vitro complex of TbpA-TbpB bound to holo human transferrin (hTf) and this triple complex was characterized by negative stain single particle electron microscopy. From the TbpA-hTf crystal structure and the TbpB-hTf SAXS structure, we made a model for the triple complex which fits beautifully into the EM 3D reconstruction. From all of these data, we now understand the specificity of recognition and have already designed antibodies that can block the TbpA-transferrin interaction. Since TbpA and TbpB are present in all clinical isolates, they represent important therapeutic targets. This is one of the largest outer membrane protein complexes characterized at high resolution and it has direct translational applications. A manuscript describing the crystallography, SAXS, EM, and functional analysis was recently submitted to Nature. Update for 2012: This work was published as an article in Nature in February 2012. We have now provided a complete structural description of the iron import pathway in Neisseria and established several testable hypotheses for iron extraction and import. This work is already regarded as a landmark paper that has significantly impacted the field and changed the course of future experiments in Neisseria.