Protein Structure, Stability, and Amyloid Formation

前列环素-1(PG-1)是一个18个残基、富含半胱氨酸的β-折叠抗菌肽(AMP)。PG-1在细胞膜上具有很强的细胞毒活性，是一种有效的抗菌剂。早些时候，我们报道了它的细胞毒性是由它的通道形成能力介导的。在这项研究中，我们研究了PG-1与定义明确的淀粉样β淀粉样蛋白(Aβ(1-42))肽的淀粉样原纤维形成特性。我们使用原子力显微镜(AFM)和硫代黄素-T染色来研究PG-1纤维生长的动力学，并用分子动力学模拟来阐明其潜在的机制。PG-1在高亲水性表面(云母)上的AFM图像显示，纤维的形态与Abeta(1-42)纤维相似。纤维生长的实时AFM成像表明，与Abeta(1-42)纤维相比，PG-1纤维的生长遵循相对较快的动力学。AFM结果与硫代黄素-T染色结果基本一致。此外，结果表明PG-1在溶液中形成了纤维。值得注意的是，相比之下，我们在阴离子脂双层2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine；上没有检测到PG-1的纤维结构，只能观察到小的PG-1低聚物。分子动力学模拟能够确定这些小分子齐聚物在膜双层上的存在。因此，我们目前的结果表明，细胞毒性AMP PG-1是淀粉样变性的，能够形成纤维。总体而言，比较富含β的AMPs和淀粉样蛋白如Abeta，除了细胞毒性和淀粉样变性外，它们有一个共同的结构基序，并且正在形成通道。这些综合性质支持淀粉样多肽和富含β片层的细胞溶解AMP之间的功能关系，表明淀粉样蛋白通道可能具有抗菌功能。阐明Abeta(1-40)纤维的结构在阿尔茨海默病研究中是很有意义的，因为它是设计在疾病早期针对Abeta(1-40)纤维形成的治疗方法所必需的。M35是一个关键的残基，因为它的潜在氧化和它在Abeta纤维中跨Beta链和Beta片层的强烈相互作用。实验上，Abeta(9-40)纤维的三重对称结构的数据表明，通过纤维轴上的M35相互作用和不同的交叉β单元之间强烈的I31-V39相互作用，形成了紧密的疏水核心。在此，我们在实验数据的基础上，探索了全长Abeta(1-40)具有三重对称性的构象。我们基于ss核磁共振数据在显式溶剂中进行的全原子分子动力学模拟重现了M35-M35和I31-V39距离的实验观察。我们对实验数据的解释表明，在纤维三重对称结构中观察到的5-7 Angstrom M35-M35距离可能与沿纤维轴的M35相互作用有关，而不是跨纤维轴的M35相互作用，因为我们测量的M35-M35跨纤维轴的距离一直高于15埃。因此，我们揭示了独特的Abeta(1-40)三角形结构沿着纤维轴有一个大的空腔，N-末端可以通过与U-转向域或C-末端结构域相互作用来帮助纤维的稳定。我们的发现，加上最近对Abeta(1-42)纤维中空核心的CyroEM表征，指出了Abeta(1-40/1-42)低聚物中空洞的相关性，当针对低聚物毒性时，应该考虑到这一点。我们阐述了转录因子(TF)沿着增强子DNA特异性组装的机制。干扰素-β增强体提供了一个很好的模型系统：它很小，其成分晶体结构可用，并且有生化和细胞数据。在干扰素-β增强体中，尽管连续的DNA反应元件(RES)重叠，但蛋白质之间的相互作用很少。我们对增强体不同基序组合的分子动力学(MD)模拟表明，协作性是通过RES的独特组织实现的：一种TF的特定结合可以通过RES的重叠来增强另一种TF与邻近RE的结合，并限制其他TF；RES的顺序可以决定形成哪些复合体；共识和非共识RES的交替可以通过优化伙伴之间的相互作用来调节结合特异性。我们的观察提供了一种解释，尽管相邻的TF之间在增强子DNA上的相互作用有限，但如何获得特异性和协同性。到目前为止，当解决选择性TF结合时，人们的注意力主要集中在RE序列上。然而，DNA上RES的顺序和它们之间的间隔物的长度可能是TF在增强子上的特定组合组装的关键因素，从而影响其功能。我们的结果强调了通过RE结合位点组织的协作性。

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich beta-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-beta (Abeta(1-42)) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Abeta(1-42) fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Abeta(1-42) fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing beta-rich AMPs and amyloids such as Abeta, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and beta-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function. Elucidating the structure of Abeta(1-40) fibrils is of interest in Alzheimer's disease research because it is required for designing therapeutics that target Abeta(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across beta-strands and across beta-sheets in Abeta fibrils. Experimentally, data for the three-fold symmetry structure of the Abeta(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-beta units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Abeta(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed 5-7 Angstrom M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 Angstrom. Consequently, we revealed that the unique Abeta(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Abeta(1-42) fibrils, point to the relevance of a cavity in Abeta(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity. We addressed the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA. The IFN-beta enhanceosome provides a good model system: it is small; its components crystal structures are available; and there are biochemical and cellular data. In the IFN-beta enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization.