Targeting protein-DNA interactions in prokaryotic systems

许多结构研究已经证实了Sigma54与RNAP和DNA的结合。Sigma54从转录起始点与DNA-24和-12碱基对结合，启动DNA融化，这是转录过程中的一个重要步骤。大量的工作，包括DNA足迹，丙氨酸扫描突变研究，Sigma54-RNA聚合酶以封闭构型与DNA结合的低分辨率冷冻电子显微镜，以及高分辨率核磁共振结构表明Sigma54与DNA的主槽特异性地紧密结合。例如，Wemmer实验室使用核磁共振技术表明，Aquifex aeolicus中的66个氨基酸的长螺旋-转角螺旋(HTH)基序与DNA结合。在这个C-末端HTH基序中，第377至386位残基的单个α螺旋(ARRTVAKYRE)被称为RpoN盒，负责与DNA的主槽结合(PBD：208K)。用丙氨酸取代Arg378、Arg379、Tyr384和Arg385可显著减少DNA结合。蛋白质的核磁共振结构表明，该螺旋与启动子的-24区(5‘-TGGCACG-3’)选择性地相互作用。特别是，Arg378和Arg379定位于相互作用的-24元素，并基于15N-HSQC谱的显著线宽，与DNA非编码链上的GUA-25和GUA-26发生多重氢键和离子相互作用。这导致了66-聚HTH基序与启动子区域(Kd=114 nM)之间的高亲和力相互作用，尽管多肽被建模为抑制转录，但有必要评估细胞活性，以确保下游的抗毒力效应是可观察到的。采用标准肉汤微量稀释法，对所有装订多肽类似物的抗菌活性进行了评价。所有多肽的最小抑菌浓度(MIC)均为32微克/毫升或更高。由于与许多常规抗生素相比，该值较高，可以在低浓度下评估该肽，以研究其对革兰氏阴性细菌毒力特性的影响。为了确定这些化合物是否对真核细胞具有细胞毒性，WS1成纤维细胞被暴露于每种化合物的连续稀释中。数据表明，在10微米的浓度范围内，这些化合物对正常细胞没有毒性。通过将化合物溶解于水中，进行了圆二色谱分析，以确定合成多肽的螺旋结构。虽然野生型sigma54多肽是非结构化的，但装订的多肽1-4都具有α-螺旋二级结构的特征谱。流式细胞术被用来高通量地观察每个装订的多肽在大量细菌种群中的有效性。通过利用其单个事件检测，细胞摄取多肽的百分比被确定。在革兰氏阴性杆菌的大肠杆菌和铜绿假单胞菌中，所有四种多肽类似物都能够穿透。与载体对照相比，野生型多肽在PA01铜绿假单胞菌中的穿透较少，在BW25113大肠杆菌中的穿透程度不显著。对于每个多肽，每个多肽样本中至少有50%的细胞被渗透。特别是，sigma54-2的外显率最高，而sigma54-4的外显率最低。为了确定细胞内的运输方式，叠氮化钠被用来灭活用于氢离子运输的ATPase。所有内部运输将通过被动或促进扩散等非主动方式进行。在这两种处理方法中，都有可能确定装订多肽的摄取模式是通过主动还是被动运输。此外，在被动转运的情况下，由于缺乏促进细胞进入所需的代谢手段，多肽的进入速度应该比主动快。在叠氮钠治疗下，大多数多肽类似物的渗透性增加。这可能是一个迹象，表明多肽能够被细菌在一定程度上泵出。总体而言，这项测试表明，与未装订的多肽相比，装订的多肽能够以一种不依赖电荷的方式更好地穿透细胞。对共聚焦显微镜进行了进一步的研究，以获得一种评估细胞外透性的低通量目视方法。这些多肽与流式细胞仪数据的相关性在于，一些细胞表现出较强的荧光强度，而另一些细胞表现出较弱的荧光强度。通过使用基于图像的通透性检测方法，我们观察到大肠杆菌的双链DNA似乎聚集在远离膜的中心。在观察到的许多细胞中，许多细胞正在进行细胞分裂，但仍大量摄取这些多肽。在细胞膜上，可以看到，由于缺乏绿色或掩盖了红膜的染色，多肽并不能自我整合到膜上。这些多肽必须穿过细胞壁和细胞膜才能留在细菌的细胞质中。通过DNA酶保护实验检测了sigma54RpoN与glnA-24位点的结合，结果表明，装订的sigma54肽可以保护DNA不被降解。为了确定这些多肽是否阻止了氮代谢基因的转录，在缺氮条件下生长的大肠杆菌细胞被装订的多肽处理。提取RNA，分析Sigma 54依赖基因glnA、yeaG和nac。我们还检测了PSPA，尽管它是一个依赖于sigma54的基因，但对氮素耗竭不敏感。结果表明，Sigma 54-2和Sigma-3在阻断依赖Sigma 54的氮耗竭反应基因转录方面效果最好。PSPA未见效果。综上所述，这些结果表明，装订的多肽可以设计成针对细菌系统以及具有很高特异性的蛋白质-DNA相互作用。

A number of structural studies have confirmed the binding of sigma54 to RNAP and DNA. Sigma54 binds DNA -24 and -12 base pairs from the transcription start site to initiate DNA melting, an essential step in transcription. A substantial body of work, including DNA footprinting, alanine scanning mutation studies, low resolution cryoelectron microscopy of sigma54-RNA-polyermase bound to DNA in the closed configuration, and high resolution NMR structure has shown that sigma54 binds specifically and tightly to the major groove of DNA. For example the Wemmer lab used NMR to show that the 66 amino acid long helix-turn-helix (HTH) motif from in Aquifex aeolicus binds DNA. Within this C-terminal HTH motif, a single alpha-helix at residues 377 to 386 (ARRTVAKYRE), termed the RpoN box, is responsible for binding to the major groove of DNA (PBD: 2O8K). Replacement of Arg378, Arg379, Tyr384 and Arg385 with Ala decreased DNA binding substantially. The protein NMR structure shows that this helix interacts selectively with the -24 region (5'-TGGCACG-3') of the promoter. In particular, Arg378 and Arg379 are localized to the -24 element of the interaction and make multiple hydrogen bonding and ionic interactions with Gua-25 and Gua-26 on the non-coding strand of DNA based on significant line-broadening of a 15N-HSQC spectrum. This leads to a high affinity interaction between the 66-mer HTH motif and the promoter region (Kd = 114 nM) Although the peptides are modeled to inhibit transcription, it is necessary to assess cell viability to ensure that downstream antivirulence effects are observable. By using the standard broth microdilution method,24 the antimicrobial activity of all stapled peptide analogs was assessed. The minimum inhibitory concentration (MIC) value of all peptides is 32 microg/mL or higher. As this value is high compared to many conventional antibiotics, the peptide can be assessed at low concentrations to study its effects on virulence properties of Gram negative bacteria. To determine whether the compounds are cytotoxic to eukaryotic cells, WS1 fibroblasts were exposed to serial dilutions of each compound. The data demonstrate that the compounds are not toxic to normal cells over a concentration range peaking at 10 microM. In order to assess the helical structure of the synthesized peptides, circular dichroism spectroscopy was carried out by dissolving the compounds in water. While the wild type sigma54 peptide is unstructured, stapled peptides 1-4 all possess the hallmark spectrum of an alpha-helical secondary structure. Flow cytometry was used to gain a high-throughput perspective on the effectiveness of each stapled peptide in a large population of bacteria. By making use of its individual event detection, the percentage of cellular uptake of the peptides was determined. In both E. coli and P. aeruginosa species of Gram negative bacteria, all four peptide analogs are capable of penetration. Compared to the vehicle control, the wild type peptide, the sequence in which no staple is present, shows minor penetration in PA01 P. aeruginosa and insignificant levels of penetration in BW25113 E. coli. With each peptide, there is a penetration of at least 50% of cell in each peptide sample. In particular, sigma54-2 appears to have the highest penetrance whereas sigma54-4 has the least. To determine the mode of transport within the cell, sodium azide was used to inactivate ATPases used in hydrogen ion transport. All interior transport will be conducted through a non-active method such as passive or facilitated diffusion. With both types of treatments, it's possible to determine whether the mode of uptake of stapled peptides is through active or passive transport. Furthermore, with passive transport, the entry of the peptide should be faster than active due to the lack of metabolic means necessary to promote cell entry. The majority of peptide analogs increase in penetration upon sodium azide treatment. This may be an indicator that the peptide is able to be pumped out by bacteria to a degree. Overall, this assay demonstrates that stapled peptides are capable of penetrating cells better than their unstapled counterpart in a charge-independent manner. Further studies in confocal microscopy were conducted to gain a low-throughput visual method of assessing cell penetrance. The peptides correlate with the flow cytometry data in that some cells display strong fluorescence intensity and others display less. By using an image-based detection method for permeability, we observe that the double-stranded DNA of E. coli appears to aggregate in the center away from the membrane. In many of the cells observed, a number of the cells were undergoing cell division but still had significant uptake of these peptides. With the cell membranes, it is seen that the peptide does not integrate itself into the membrane due to a lack of green or masking of the red membrane stain. These peptides must cross both the cell wall and cell membrane to remain in the cytoplasm of bacteria. The binding of sigma54 RpoN with the -24 site of glnA was examined by performing a DNase protection assay which showed that stapled sigma 54 peptides protected DNA from degradation. To determine whether the peptides blocked the transcription of nitrogen metabolism genes, E. coli cells grown under nitrogen deficient conditions were treated with stapled peptides. RNA was isolated, and the sigma 54 dependent genes glnA, yeaG, and nac were analyzed. We also examined pspA which, despite being a sigma54 dependent gene, is insensitive to nitrogen depletion. The data showed that stapled sigma 54-2 and -3 were the best at blocking the transcription of sigma 54-dependent nitrogen depletion response genes. No effects were seen with pspA. Taken together, these results demonstrate that stapled peptides can be designed to target bacterial systems as well as protein-DNA interactions with a great level of specificity.