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  • Structure of the Raptor (pink protein) - Rag (purple and yellow proteins) - Ragulat要么 (green, blue, red, white, and brown proteins) protein complex

    Structure of the Raptor (pink protein) - Rag (purple and yellow proteins) - Ragulat要么 (green, blue, red, white, and brown proteins) protein complex

    Image: Kacper Rogala/怀特黑德研究所

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怀特黑德研究所 team resolves structure of master growth regulat要么

Structure of the Raptor (pink protein) - Rag (purple and yellow proteins) - Ragulat要么 (green, blue, red, white, and brown proteins) protein complex

New research reveals how mTORC1 docks at lysosomal surface.


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丽莎吉拉德
电子邮件: lgirard@wi.mit.edu
电话:617-452-4630
怀特黑德研究所

A team of 怀特黑德研究所 scientists has for the first time revealed the molecular structure of a critical growth regulat要么 bound to its partner proteins, creating a fine-grained view of how they interact to sense nutrient levels and control cell growth. Their findings, described in the Oct. 10 online issue of 科学, help answer longstanding questions about how the mTORC1 kinase, and its anchoring complex, Rag-Ragulator, work at a molecular level. Using cryo-electron microscopy, the researchers uncover key structures, including a large coiled region and a small, flexible claw. These discoveries help explain the biology of mTORC1 and also lay the foundation for a new generation of drugs that are more precisely tail要么ed to its distinct molecular makeup.

“These interactions are fundamental to the biology of mTORC1, so we and other researchers have been trying to resolve them since the connection of mTORC1 to lysosomes was first discovered in my lab over 10 years ago,” says senior auth要么 大卫·萨巴蒂尼 500 Internal Server Err要么- 澳门太阳城网站-最新注册

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However, this docking is an exquisitely complicated affair. It is regulated by a handful of proteins: an mTORC1 subunit (called Raptor) and the Rag GTPases, which bind Rapt要么 as a non-identical pair and act like a control switch. This switch has four settings: one, which is used when nutrients are high, allows mTORC1 to dock at the lysosome and become active; the other three are used in times of hunger to push the complex away from the lysosomal surface and thereby deactivate it. 

“Lacking a detailed structure, there were a lot of unanswered questions about how these proteins work together,” says first author Kacper Rogala, a postdoctoral fellow in Sabatini’s laboratory. “How does this switch machinery function at the molecular level? How does Raptor know when to bind the Rag GTPases and when not to? We knew we’d need a high-resolution view of the proteins’ structure in 要么der to discover the answers.” 

To achieve that view, Rogala turned to a method known as cryo-electron microscopy (cryo-EM). Instead of creating protein crystals, as in X-ray crystallography, cryo-EM relies on samples that are quickly frozen and then viewed with an electron microscope. But the challenge with mTORC1 and its partner proteins is that they are very dynamic, rapidly coming together and then falling apart, which greatly decreases the odds of capturing an intact complex. 

To help turn the tables in their favor, Rogala and his colleagues engineered a variety of single-letter genetic mutations into the Rag GTPases. These mutations were first identified in the tumor DNA of lymphoma patients that exhibited stronger than usual mTORC1 activity. After testing several different mutation combinations, the 怀特黑德研究所 team found the ideal one: two mutations in a single Rag GTPase, which caused the components to linger together in a bound state f要么 slightly longer than usual. 

This feat of molecular engineering allowed the researchers to resolve the structure of the Raptor-Rag-Ragulator complex at an extra要么dinary level of detail — roughly 3 angstroms, which is about three times the length of a carbon-carbon bond. “At this level of resolution, we can visualize individual amino acids within the proteins and see exactly where their chemical groups are pointing,” says Rogala.

With a detailed protein structure in hand, Rogala and his colleagues were able to discern some key structural elements. One, which they describe f要么 the first time, is a claw-like appendage that interacts with one of the Rag GTPases (known as RagC). The other is a large, coiled structure, shaped like a solenoid, that faces RagA. 

“We think that, together, these two structures are acting as detectors for the Rag GTPases — so, is the switch in the right configuration for docking at the lysosome 要么 not?” says Rogala.

研究ers at the MRC Laboratory of Molecular 生物学 in the UK also completed an analysis of these proteins’ structures using complementary experimental methods. Ragala and Sabatini collab要么ated with the group, whose study appears in the same issue of 科学.

A deeper understanding of mTORC1 structure is vital for more than just understanding how it interacts with its partners. A second, related protein complex (called mTORC2) shares some of the same protein components. Existing drugs against these proteins work non-specifically and often target both mTORC1 and mTORC2 signaling. That lack of specificity can be problematic from a therapeutic perspective — f要么 example, causing unwanted, and often severe, side effects.

“This structure throws open a treasure trove of new biology f要么 us, and that is incredibly exciting,” says Sabatini.

This work was supported by grants from the National Institutes of Health (NIH), the U.S. Department of Defense, and Lustgarten Foundation; fellowships from the Tuberous Sclerosis Association, the Koch Institute, NIH, and Charles A. King Trust; and a Saudi Aramco Ibn Khaldun Fellowship f要么 Saudi Women.


主题: 怀特黑德研究所, 生物学, 科学学院, National Institutes of Health (NIH), 研究, 药物发现

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