據(jù)美國癌癥協(xié)會預(yù)估，在2022年美國將有大約2萬急性髓系白血?。ˋML）新發(fā)病例。斯坦福大學(xué)醫(yī)學(xué)院教授Kathleen Sakamoto長期致力于開發(fā)針對AML和其他血液疾病的治療方法。然而，她的團(tuán)隊(duì)在研究過程中受到X射線晶體學(xué)和冷凍電鏡這兩種技術(shù)之間微妙差距的阻礙。

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.

問題的一部分出自KIX，這是CREB結(jié)合蛋白（CBP）的一個片段，AML癌細(xì)胞用它來轉(zhuǎn)錄那些有利于其生長和存活的基因。該團(tuán)隊(duì)表示，更好地了解KIX結(jié)構(gòu)可以幫助他們設(shè)計(jì)出抑制KIX并防止癌細(xì)胞復(fù)制的藥物。然而，使用X射線晶體學(xué)方法研究CREB結(jié)合蛋白的嘗試并不成功。根據(jù)晶體學(xué)標(biāo)準(zhǔn)，該蛋白的大尺寸直接導(dǎo)致其難以結(jié)晶。即使勉強(qiáng)結(jié)晶，結(jié)晶的過程也導(dǎo)致藥物設(shè)計(jì)者難以對KIX部分進(jìn)行分析和靶點(diǎn)瞄準(zhǔn)。

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本身過小，導(dǎo)致難以使用冷凍電鏡對其進(jìn)行有效研究。為了通過冷凍電鏡獲得蛋白質(zhì)的良好圖像，研究人員必須能夠在電鏡圖像中找到足夠多的蛋白質(zhì)副本，并了解它們是如何定向的。只有通過對蛋白質(zhì)圖像進(jìn)行檢索并排列，冷凍電鏡才能產(chǎn)生高分辨率結(jié)構(gòu)。按照冷凍電鏡的標(biāo)準(zhǔn)，KIX的小尺寸對于結(jié)構(gòu)解析來說是極具挑戰(zhàn)性的。另一種方法，即核磁共振，已被用來確定KIX與其他自然生成的分子結(jié)合時的結(jié)構(gòu)，但該方法需要大量的準(zhǔn)備和分析，使得它不太適合于快速確定分子的結(jié)構(gòu)，因此也不太適合于研究潛在的KIX抑制藥物的效果。

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.

"冷凍電鏡（cryo-EM）已成為針對人類疾病的分子治療開發(fā)的可行結(jié)構(gòu)工具，"研究人員寫道。"然而，解析小于30 kDa的活性蛋白結(jié)構(gòu)仍然是一個挑戰(zhàn)。CREB結(jié)合蛋白（CBP）的11 kDa KIX結(jié)構(gòu)域是急性髓系白血病和其他癌癥的潛在治療靶點(diǎn)，也是一種一直無法進(jìn)行基于結(jié)構(gòu)的抑制劑設(shè)計(jì)的蛋白質(zhì)。因此，我們開發(fā)了一種實(shí)驗(yàn)方法，通過設(shè)計(jì)蛋白質(zhì)雙殼來克服尺寸限制，將KIX結(jié)構(gòu)域夾在Apoferritin內(nèi)殼和麥芽糖結(jié)合蛋白外殼的中間。

“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.”

斯坦福大學(xué)醫(yī)學(xué)院、工程學(xué)院以及SLAC國家加速器實(shí)驗(yàn)室的研究人員已經(jīng)找到了一種方法來彌合這一差距，他們使用分子籠來穩(wěn)定某些中等大小的蛋白質(zhì)，使得它們可以進(jìn)行首次的冷凍電鏡成像，并揭示原子級的細(xì)節(jié)。他們的研究結(jié)果發(fā)表在ACS Central Science雜志上，標(biāo)題為"基于冷凍電鏡、蛋白質(zhì)工程和模擬的肽療法開發(fā)，用于對抗急性髓系白血病"。

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.”

當(dāng)研究人員將一批KIX蛋白質(zhì)夾在一個球形分子和外部的分子籠之間時，解決方案便出現(xiàn)了。這種"雙殼"比單個KIX分子大得多，也更容易在冷凍電鏡圖像中被發(fā)現(xiàn)和定向，使得獲得KIX分子本身的高分辨率圖像變得更加容易。除了看到KIX的結(jié)構(gòu)外，研究人員還能夠在混合物中添加其他分子，看看它們是否可能與KIX的功能結(jié)合并抑制KIX的功能。該團(tuán)隊(duì)報告說，他們已經(jīng)能夠使這種結(jié)合強(qiáng)度提高約200倍，這可以幫助科學(xué)家開發(fā)出在較低劑量下有效的藥物。

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.

該團(tuán)隊(duì)的研究結(jié)果還表明，這種方法對中等大小的蛋白質(zhì)也有作用，特指那些很難用冷凍電鏡或X射線晶體學(xué)進(jìn)行研究的蛋白，例如一些病毒蛋白。"我們正在向前邁進(jìn)，以擴(kuò)大該方法的適用性，"斯坦福大學(xué)SLAC中心的教授Soichi Wakatsuki博士解釋說。

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.

本文轉(zhuǎn)載自GEN News：

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

支持性內(nèi)容

Supportive content

相關(guān)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

最近在冷凍電鏡（cryo-EM）單顆粒技術(shù)的突破已經(jīng)實(shí)現(xiàn)了許多大分子的高分辨率結(jié)構(gòu)解析。對于小于40 kDa且無法通過核磁共振（NMR）結(jié)晶或成像的樣品，冷凍電鏡也難以進(jìn)行結(jié)構(gòu)解析，導(dǎo)致結(jié)構(gòu)生物學(xué)領(lǐng)域存在技術(shù)空白。為了對這些小分子量蛋白質(zhì)成像，我們已經(jīng)付出了艱辛的努力，包括優(yōu)化樣品制備及相板的應(yīng)用，以及將小分子量蛋白質(zhì)與具有已知結(jié)構(gòu)的較大分子結(jié)合起來的納米籠系統(tǒng)的設(shè)計(jì)。然而，通過冷凍電鏡在3.5 ?分辨率下測定的最小蛋白質(zhì)的分子量仍高于50 kDa。除蛋白質(zhì)外，我們還通過冷凍電鏡研究了一些小于50 kDa的小RNA，從而為40 kDa 的SAM-IV核糖開關(guān)實(shí)現(xiàn)了3.7 ?的分辨率結(jié)構(gòu)。然而，迄今為止，還沒有低于40 kDa的多肽能通過冷凍電鏡單顆粒技術(shù)實(shí)現(xiàn)4 ?分辨率的結(jié)構(gòu)解析。本研究表明，將小分子量蛋白質(zhì)融合為籠狀結(jié)構(gòu)是打破冷凍電鏡結(jié)構(gòu)測定的分辨率極限的可行方案。

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 ? 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 ? resolution structure for a 40 kDa SAM-IV riboswitch.??However, to date, no polypeptides below 40 kDa have been resolved to better than 4 ? 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.

水木視界丨iss. 6

每周分享結(jié)構(gòu)生物學(xué)前沿進(jìn)展