Structural characterization of OM proteins from Gram-negative pathogens

我们早期的晶体结构显示了铁转运蛋白是如何识别结合到小分子上的Fe3+的，比如Enterobactin(一种由大肠杆菌合成的铁载体)和柠檬酸盐。每个转运体都有一个与其首选小分子唯一的结合口袋。当正确的底物结合时，转运蛋白经历构象变化，通过外膜发送信号，并为运输系统做好准备。 我们最近扩大了这方面的研究，以确定脑膜炎奈瑟菌是如何与人血清转铁蛋白结合并提取铁以输入细菌细胞的。这些细菌需要铁来生存，并直接从人类蛋白质中获得铁。奈瑟氏菌有一种外膜蛋白TbpA和一种共受体蛋白TbpB，这两种蛋白一起可以从一种名为转铁蛋白的人类血浆蛋白中提取铁。我们采用X射线结晶学、电子显微镜、小角X射线散射、生物化学和分子动力学模拟相结合的方法来阐明其清除铁的机理。这是细菌外膜蛋白与其全长人类目标蛋白结合的第一个原子分辨结构(Noinaj等人和Buchanan，自然，2012年)。 我们已经扩大了我们对过渡金属转运到鲍曼不动杆菌锌摄取的关注。鲍曼不动杆菌是一种医院获得性感染，表现出多重耐药性。NIH临床中心对此非常感兴趣。锌与鲍曼不动杆菌的毒力相关，该细菌中存在三种可能依赖于TonB的锌转运蛋白。当缺乏锌时，鲍曼不动杆菌对现有的抗生素变得更加敏感，因此抑制锌的摄取可能会导致针对这种革兰氏阴性菌的新疗法。我们刚刚解决了一种结合锌的鲍曼不动杆菌锌转运蛋白的结构。我们正在利用该结构进行电子小分子药物筛选，并研究锌的转运途径。一份描述这部作品的手稿正在准备中。 有趣的是，对于所有这些过渡金属转运体来说，金属是如何进入周质的并不是很清楚。我们知道，运输涉及到内膜蛋白复合体(TonB-ExbB-ExbD)和以质子动力形式存在的能量。我们目前正在努力提供有关运输过程的结构性数据。 在我们寻找新的抗菌疗法的过程中，我们扩展了我们在小分子转运体方面的工作，以研究蛋白质是如何通过外膜运输的。我们研究的一些铁转运蛋白也有助于吸收被称为结肠素的大蛋白毒素。例如，我们确定了鼠疫耶尔森氏菌(引起鼠疫)的外膜铁转运体的结构，这是毒力所需的。我们还确定了一种名为Posticin的粘菌素的结构，它使用这种转运蛋白穿过外膜。这两种结构向我们展示了如何设计一种新型的抗生素，这是针对任何革兰氏阴性细菌的第一个噬菌体疗法例子，我们的抗生素被证明对一些临床分离株有效(Lukack等人和Buchanan，2012年PNAS)。在这一成功的指导下，我们将继续对其他细菌病原体进行这种类型的蛋白质工程。 另一种对NIH临床中心非常重要的医院获得性感染是肺炎克雷伯菌。这种细菌表现出多重耐药性，一些菌株已经表现出超强毒力。为了找到对抗感染的新方法，我们正在与NCI的Susan Gottesman合作，研究与胶囊调节有关的蛋白质。肺炎克雷伯菌可以逃脱免疫检测，并通过其包裹在外膜上的厚厚的包膜层来阻止抗生素的渗透。我们的假设是，下调包膜合成可能会使肺炎克雷伯菌对现有抗生素更敏感，因此比目前的情况更容易治疗。对该系统的结构和功能实验正在进行中。 最后，除了研究外膜转运蛋白外，我们还对任何对细菌生长至关重要的外膜蛋白复合体感兴趣，因为这些蛋白可能成为很好的疫苗和药物靶标。LptDE就是这样一种复合体，它将新合成的脂多糖(LPS)放入外膜。在大多数革兰氏阴性细菌中，这种复合体是生存所必需的。我们最近从三种不同的细菌病原体那里解决了四种LptDE结构，揭示了脂多糖是如何结合到外膜上的。LptD是已知的最大的外膜蛋白之一，有26条β链。LptE位于LptD桶内，除了与脂多糖的亲水部分相互作用外，还堵塞毛孔。2016年，我们发表了三种病原体的四种LptDE结构。由于整个LPT途径在大多数革兰氏阴性菌中是必不可少的，我们正在利用这些结构来开发新的抗菌试剂。

Our early crystal structures showed how iron transporters specifically recognize Fe3+ bound to small molecules such as enterobactin (a siderophore synthesized by Escherichia coli) and citrate. Each transporter has a unique binding pocket for its preferred small molecule. When the correct substrate binds, the transporter undergoes conformational changes that send a signal across the outer membrane and prepare the system for transport. We recently expanded our studies in this area to determine how Neisseria meningitidis binds to human serum transferrin and extracts the iron for import into the bacterial cell. These bacteria require iron for survival and obtain it directly from human proteins. Neisseria have an outer membrane protein, TbpA, and a co-receptor protein, TbpB, which together can extract the iron from a human plasma protein called transferrin. We used a combined approach of X-ray crystallography, electron microscopy, small angle X-ray scattering, biochemistry, and molecular dynamics simulations to elucidate the iron-scavenging mechanism. This was the first atomic resolution structure of a bacterial outer membrane protein bound to its full-length human target protein (Noinaj et al and Buchanan, Nature 2012). We have expanded our focus on transition metal transport to Acinetobacter baumanni zinc uptake. A. baumannii is a hospital-acquired infection demonstrating multidrug resistance. It is of great interest to the NIH clinical center. Zinc correlates with virulence in A. baumannii and there are three putative TonB-dependent zinc transporters in this bacterium. When deprived of zinc, A. baumannii becomes much more sensitive to existing antibiotics, so inhibition of zinc uptake may lead to novel therapies against this Gram-negative bacterium. We just solved the structure of an A. baumannii zinc transporter with zinc bound. We are using the structure to perform in silico small molecule drug screening and to investigate the zinc transport pathway. A manuscript describing this work is in preparation. Interestingly, for all of these transition metal transporters, how the metal gets into the periplasm is not well understood. We know that transport involves an inner membrane protein complex (TonB-ExbB-ExbD) and energy in the form of protonmotive force. We are currently working to provide structural data on the transport process. In our search for novel antimicrobial therapies, we extended our work on small-molecule transporters to ask how proteins are ferried across the outer membrane. Some of the iron transporters that we study also facilitate the uptake of large protein toxins called colicins. For example, we determined the structure of an outer membrane iron transporter from Yersinia pestis (which causes plague) that is required for virulence. We also determined the structure of a colicin, called pesticin, which uses this transporter to cross the outer membrane. The two structures showed us how to engineer a novel antibiotic that is the first example of phage therapy for any Gram-negative bacterium, and our antibiotic was demonstrated to be effective on a number of clinical isolates (Lukacik et al and Buchanan, PNAS 2012). Guided by this success, we will continue this type of protein engineering for other bacterial pathogens. Another hospital-acquired infection of great importance to the NIH clinical center is Klebsiella pneumoniae. This bacterium exhibits multidrug resistance and some strains have shown hypervirulence. In an effort to identify new ways to combat infection, we are collaborating with Susan Gottesman, NCI, to investigate proteins involved in regulation of capsule. K. pneumoniae can escape immune detection and prevent penetration of antibiotics with its thick capsule layer that surrounds the outer membrane. Our hypothesis is that down-regulation of capsule synthesis might make K. pneumoniae more sensitive to available antibiotics, and thus more treatable than is currently the case. Structural and functional experiments on this system are in progress. Finally, in addition to studying outer membrane transporters, we are interested in any outer membrane protein complexes that are essential for bacterial growth, since these proteins may make good vaccine and drug targets. One such complex is LptDE, which puts newly synthesized lipopolysaccharide (LPS) into the outer membrane. In most Gram-negative bacteria, this complex is essential for viability. We recently solved four LptDE structures from three different bacterial pathogens, shedding light on how lipopolysaccharide is incorporated into the outer membrane. LptD is one of the largest outer membrane proteins known, with 26 beta strands. LptE sits inside the LptD barrel and occludes the pore, in addition to interacting with the hydrophilic portion of LPS. We published four LptDE structures from three pathogens in Structure in 2016. Since the entire Lpt pathway is essential in most Gram-negative bacteria, we are using these structures to develop new antimicrobial reagents.