本文转载自GEN News：

"Cryo-EM Provides New Insights into Acute Myeloid Leukemia"

据美国癌症协会预估，在2022年美国将有大约2万急性髓系白血病（AML）新发病例。斯坦福大学医学院教授Kathleen Sakamoto长期致力于开发针对AML和其他血液疾病的治疗方法。然而，她的团队在研究过程中受到X射线晶体学和冷冻电镜这两种技术之间微妙差距的阻碍。

The American Cancer Society estimates there will be about 20,050 new cases of acute myeloid leukemia (AML) in the United States in 2022. Stanford School of Medicine professor Kathleen Sakamoto, MD, PhD, has been working on the development of therapeutics against AML and other blood disorders. Her team, however, was hampered in their search by a subtle gap between two technologies, X-ray crystallography and cryogenic electron microscopy.

左：显微镜下健康的骨髓

右：急性髓系白血病(AML)患者的骨髓

Left: Healthy bone marrow appears through a microscope

Right: bone marrow of a pediatric patient with acute myeloid leukemia

问题的一部分出自KIX，这是CREB结合蛋白（CBP）的一个片段，AML癌细胞用它来转录那些有利于其生长和存活的基因。该团队表示，更好地了解KIX结构可以帮助他们设计出抑制KIX并防止癌细胞复制的药物。然而，使用X射线晶体学方法研究CREB结合蛋白的尝试并不成功。根据晶体学标准，该蛋白的大尺寸直接导致其难以结晶。即使勉强结晶，结晶的过程也导致药物设计者难以对KIX部分进行分析和靶点瞄准。

A part of the issue is KIX, a segment of the CREB Binding Protein (CBP) that AML cancer cells use to transcribe genes important for growth and survival. The team say that understanding its structure better could enable them to design drugs that inhibit KIX and prevent cancer cells from replicating. However, efforts to study the protein using X-ray crystallography have not been successful; the molecule’s generous size, by crystallography standards, makes it harder to crystallise and even when it has been crystallised, the particulars of that process have made it harder to analyse the parts of KIX that drug designers would like to target.

此外，KIX本身过小，导致难以使用冷冻电镜对其进行有效研究。为了通过冷冻电镜获得蛋白质的良好图像，研究人员必须能够在电镜图像中找到足够多的蛋白质副本，并了解它们是如何定向的。只有通过对蛋白质图像进行检索并排列，冷冻电镜才能产生高分辨率结构。按照冷冻电镜的标准，KIX的小尺寸对于结构解析来说是极具挑战性的。另一种方法，即核磁共振，已被用来确定KIX与其他自然生成的分子结合时的结构，但该方法需要大量的准备和分析，使得它不太适合于快速确定分子的结构，因此也不太适合于研究潜在的KIX抑制药物的效果。

Furthermore, KIX is too small on its own to study effectively with cryo-EM. To get good images of a protein with cryo-EM, researchers must be able to locate enough copies of the protein within an electron microscope image, then understand how they are oriented. Only by finding and lining up images of a protein can cryo-EM methods yield high-resolution structures. KIX’s small size, by cryo-EM standards, makes that a challenge. Another option, nuclear magnetic resonance, has been used to determine the structure of KIX when bound to other naturally occurring molecules, but the method requires extensive preparation and analysis, making it less than ideal for quickly determining molecule’s structures and therefore for less than ideal for studying the effects of potential KIX-inhibiting drugs.

"冷冻电镜（cryo-EM）已成为针对人类疾病的分子治疗开发的可行结构工具，"研究人员写道。"然而，解析小于30 kDa的活性蛋白结构仍然是一个挑战。CREB结合蛋白（CBP）的11 kDa KIX结构域是急性髓系白血病和其他癌症的潜在治疗靶点，也是一种一直无法进行基于结构的抑制剂设计的蛋白质。因此，我们开发了一种实验方法，通过设计蛋白质双壳来克服尺寸限制，将KIX结构域夹在Apoferritin内壳和麦芽糖结合蛋白外壳的中间。

“Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases,” the researchers wrote. “However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein that has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell.”

双壳系统的设计

Design of the double-shell system

斯坦福大学医学院、工程学院以及SLAC国家加速器实验室的研究人员已经找到了一种方法来弥合这一差距，他们使用分子笼来稳定某些中等大小的蛋白质，使得它们可以进行首次的冷冻电镜成像，并揭示原子级的细节。他们的研究结果发表在ACS Central Science杂志上，标题为"基于冷冻电镜、蛋白质工程和模拟的肽疗法开发，用于对抗急性髓系白血病"。

Researchers at Stanford University’s Schools of Medicine and Engineering and the Department of Energy’s SLAC National Accelerator Laboratory have found a way to bridge that gap by using a kind of molecular cage to stabilize certain medium-sized proteins so they can be imaged for the first time with cryo-EM, which can reveal almost atomic-level details.Their findings are published in the journal ACS Central Science in a paper titled, “Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia.”

当研究人员将一批KIX蛋白质夹在一个球形分子和外部的分子笼之间时，解决方案便出现了。这种"双壳"比单个KIX分子大得多，也更容易在冷冻电镜图像中被发现和定向，使得获得KIX分子本身的高分辨率图像变得更加容易。除了看到KIX的结构外，研究人员还能够在混合物中添加其他分子，看看它们是否可能与KIX的功能结合并抑制KIX的功能。该团队报告说，他们已经能够使这种结合强度提高约200倍，这可以帮助科学家开发出在较低剂量下有效的药物。

The solution came to the researchers when they would sandwich batches of KIX proteins between a central, ball-shaped molecule and an outer molecular cage. Because this “double shell” was much larger than individual KIX molecules, it would be easier to spot and orient in cryo-EM images, and that would make it easier to get high-resolution images of the KIX molecules themselves. In addition to seeing KIX’s structure, the researchers were able to add other molecules to the mix to see if they might bind to and potentially inhibit KIX’s function. Already, the team reports, they have been able to make that bonding about 200 times stronger, which could help scientists develop drugs that are effective at lower doses.

夹在MBP外壳（淡紫色）和去铁蛋白内壳（青色）之间的KIX（洋红色）的冷冻电镜重构。这使得研究人员获得了迄今为止最好的KIX外观，KIX是治疗急性髓系白血病的潜在靶点。

Cryo-EM reconstructions of KIX (red) sandwiched between an MBP outer shell (purple) and an apoferritin inner shell (blue). The sandwiching technique helped researchers get the best look yet of KIX, a potential target for treating acute myeloid leukaemia. 

[Credit: SLAC National Accelerator Laboratory.]

接上图，更细节的冷冻电镜重构：中央去铁蛋白（青色）和周围的 KIX 蛋白（洋红色)， MBP外壳并未显示。

A more detailed cryo-EM reconstruction of KIX proteins (magenta) surrounding the central apoferritin shell (cyan). The outer MBP shell is not shown. 

(Greg Stewart/SLAC National Accelerator Laboratory)

该团队的研究结果还表明，这种方法对中等大小的蛋白质也有作用，特指那些很难用冷冻电镜或X射线晶体学进行研究的蛋白，例如一些病毒蛋白。"我们正在向前迈进，以扩大该方法的适用性，"斯坦福大学SLAC中心的教授Soichi Wakatsuki博士解释说。

The team’s results also suggest this method could prove useful for other proteins of in-between sizes that are hard to study with either cryo-EM or X-ray crystallography, including, perhaps, some viral proteins. “We are moving forward to expand the applicability of the approach,” explained Soichi Wakatsuki, PhD, SLAC and Stanford professor.

支持性内容

Supportive content

相关ACS Central Science文章: 

“Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia.”, DOI: 10.1021/acscentsci.1c01090

最近在冷冻电镜（cryo-EM）单颗粒技术的突破已经实现了许多大分子的高分辨率结构解析。对于小于40 kDa且无法通过核磁共振（NMR）结晶或成像的样品，冷冻电镜也难以进行结构解析，导致结构生物学领域存在技术空白。为了对这些小分子量蛋白质成像，我们已经付出了艰辛的努力，包括优化样品制备及相板的应用，以及将小分子量蛋白质与具有已知结构的较大分子结合起来的纳米笼系统的设计。然而，通过冷冻电镜在3.5 Å分辨率下测定的最小蛋白质的分子量仍高于50 kDa。除蛋白质外，我们还通过冷冻电镜研究了一些小于50 kDa的小RNA，从而为40 kDa 的SAM-IV核糖开关实现了3.7 Å的分辨率结构。然而，迄今为止，还没有低于40 kDa的多肽能通过冷冻电镜单颗粒技术实现4 Å分辨率的结构解析。本研究表明，将小分子量蛋白质融合为笼状结构是打破冷冻电镜结构测定的分辨率极限的可行方案。

Recent technological breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have achieved numerous high-resolution structures of macromolecules. For specimens smaller than 40 kDa that cannot be crystallized or imaged by nuclear magnetic resonance (NMR), cryo-EM is also difficult to apply, leading to a big gap in the field of structural biology. Extensive efforts have been made to visualize small proteins, including optimization of sample preparation, application of a phase plate, and the design of nanocage systems that link the small proteins to larger molecules with a known structure. However, the molecular weight of the smallest protein determined by cryo-EM at better than 3.5 Å resolution is still higher than 50 kDa.  Besides proteins, some small RNAs less than 50 kDa have been studied by cryo-EM, achieving a 3.7 Å resolution structure for a 40 kDa SAM-IV riboswitch.  However, to date, no polypeptides below 40 kDa have been resolved to better than 4 Å by single-particle cryo-EM. This study demonstrates that the fusion of small proteins to a cage-like structure is a feasible solution to break the resolution limit for cryo-EM structural determination of small biomolecules.

上图左侧，两个相邻亚基在两个不同视图中的冷冻电镜密度。

Left, cryo-EM density of two adjacent subunits in two different views. 

上图右侧，放大视图显示KIX和去铁蛋白之间的两个二硫键，并注释了三个螺旋的位置。

Right, zoomed view to show the two disulfide bonds between KIX and apoferritin, and the positions of three helices are annotated.

如上图所示，双壳设计允许荧光偏振测定，确认双壳中的KIX结构域与这些相互作用的肽之间的结合。进一步的冷冻电镜分析揭示了单个KIX螺旋和最佳肽之间的螺旋间相互作用，为下一代抑制剂的开发提供了可行的思路。

The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix–helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.

水木视界丨iss. 6

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