Author: Raleigh McElvery

Whitehead Institute

October 10, 2019

Cambridge, MA — A team of Whitehead Institute scientists has for the first time revealed the molecular structure of a critical growth regulator 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 October 10th online issue of Science, 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 tailored 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 author David Sabatini, a Member of Whitehead Institute, a professor of biology at Massachusetts Institute of Technology, and investigator with the Howard Hughes Medical Institute (HHMI). “Now, we have a really deep look at how this important complex works, which opens up a panorama of new research.”

mTORC1 is a massive protein complex that enables cells to respond appropriately when food is either abundant or scarce, and has been implicated in a wide range of human diseases, including cancer, diabetes, and neurodegenerative disease. It operates within tiny compartments known as lysosomes — miniature recycling stations of the cell. In order to sense nutrient levels in the lysosome, and become active, mTORC1 must first dock at the lysosomal surface, where it meets up with its anchoring protein (called Rag-Ragulator).

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 Raptor 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 order to discover the answers.”

To achieve that view, Rogala turned to a method known as cryo-electron microscopy or 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 Whitehead Institute team found the ideal one: two mutations in a single Rag GTPase, which caused the components to linger together in a bound state for slightly longer than usual.

This feat of molecular engineering allowed the researchers to resolve the structure of the Raptor-Rag-Ragulator complex at an extraordinary 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 for 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 or not?” says Rogala.

Researchers at the MRC Laboratory of Molecular Biology in the UK also completed an analysis of these proteins’ structures using complementary experimental methods. Rogala and Sabatini collaborated with the group, whose study appears in the same issue of Science.

A deeper understanding of mTORC1 structure is vital not just for 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 — for example, causing unwanted, and often severe, side effects.

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

This work was supported by grants from the NIH (R01 CA103866, R01 CA129105, and R37 AI47389), Department of Defense (W81XWH-07-0448), and Lustgarten Foundation; fellowships from the Tuberous Sclerosis Association, the Koch Institute, NIH (F30 CA236179), and Charles A. King Trust; and a Saudi Aramco Ibn Khaldun Fellowship for Saudi Women. David M. Sabatini is an investigator of the Howard Hughes Medical Institute and an ACS Research Professor.

Papers cited:

Rogala K.B. et al. Structural basis for the docking of mTORC1 on the lysosomal surface. Science. DOI: 10.1126/science.aay0166

Madhanagopal et al. Architecture of human Rag GTPase heterodimers and their complex with mTORC1. Science. DOI: 10.1126/science.aax3939

Jeandele Elliot spent the summer studying a durable compound in pollen and developing equally durable friendships.

Saima Sidik

September 9, 2019

Jeandele Elliot was raised on poetry. Like the Nobel Prize winning writer Derek Walcott, she grew up on the Caribbean island of St. Lucia where the locals celebrate the epic, multi-volume poems that won Walcott the 1992 prize for literature. Elliot grew up with the adults around her extolling Walcott’s brilliance, but it wasn’t until she left St. Lucia that she understood why this island was so inspirational to Walcott. Young Walcott left St. Lucia to pursue a life as a writer decades before Elliot was born; similarly, Elliot left to study chemical engineering at Howard University in Washington, D.C. Although their vocations differ, Walcott infused his work with the qualities of his home country before his death in 2017, just as Elliot does today. While Walcott’s poetry returns again and again to his love for the island’s people and natural landscape, Elliot applies St. Lucia’s culture of hard work and resilience to her science.

These traits have served Elliot well at Howard University, where she’s currently entering her junior year. It also earned her a spot in MIT’s Summer Research Program in Biology (MSRP-Bio), for which she received a scholarship from the Gould Fund. During this 10-week internship, Elliot worked in biology professor Jing-Ke Weng’s lab, studying the biochemical pathway that produces sporopollenin, an exceptionally strong substance that coats and protects pollen grains.

Elliot has loved science since she was in high school, and her ambition to be an impactful researcher was initially inspired by the value St. Lucians place on academic success. Walcott is one of two Nobel Laureates who grew up on St. Lucia — the second being Sir Arthur Lewis, who won the 1979 Nobel Memorial Prize in Economic Sciences — and every year the locals celebrate these two citizens during Nobel Laureate Week. The celebrations inspired Elliot to aim high when it came to her own career. “These people are from the same culture as I am, and they got so far. So I can definitely do the same thing; there’s nothing holding me back,” she says.

Elliot’s mother, a middle school principal, shared this sentiment, and she made it clear that she expected all of her children to pursue higher education. In preparation, she encouraged Elliot and her three siblings to focus on science starting in grade nine, when the St. Lucian school system requires students to begin specializing in either science, business, or arts. Scientific careers require many years of education, so she thought it would be best for her children to start learning this discipline early, even if they decided to switch to other careers later down the line.

“I studied science in high school knowing that I had to, but I also really enjoyed it,” Elliot says. She especially loved drawing chemical structures, then picturing these same structures as components of the reactions that changed colors and emitted interesting smells when the class performed experiments.

Sometimes practical considerations interfere with passions, however, and there was one hurdle Elliot had to overcome before she could attend college: money. Educating her three older siblings had exhausted her family’s finances, so Elliot was on her own when it came to figuring out how to pay for school. After finishing a two-year course of study in sciences called “A-levels” that St. Lucians pursue after high school, Elliot spent an additional two years working in a high school science lab while she looked for scholarships.

“That was one of the hardest times in my life because I wasn’t guaranteed to go to university,” she says. But, inspired by the Caribbean spirit of resilience, she resolved to find a way. In the end, Howard University offered her a full scholarship, which she happily accepted.

“Before I went to college, I had this infatuation with doing research,” Elliot says. “When I went to Howard, I was able to join a lab, and then I fully realized my passion.”

Elliot became captivated by the millimeter-long nematode Caenorhabditis elegans, and she discovered that a group of enzymes known for their role in protein degradation have a second function that affects the worm’s fertility. Not everyone would have enjoyed the hours that Elliot spent propagating tiny worms by moving them from one agar-filled petri dish to another, but she loved the moments of discovery that followed her hard work. That was when she knew she wanted to pursue a research career.

Elliot sought advice on her college applications from fellow Caribbean and MIT electrical engineering professor Cardinal Warde, who coordinates a program that introduces Carribean high school students to STEM careers. In addition to helping with her college applications, Warde told her about MSRP-Bio. This rigorous dive into research sounded like great preparation for graduate school, so the conversation stuck with Elliot, and several years later she applied, got in, and joined the Weng lab, studying sporopollenin.

Elliot spent the summer engineering bacteria to produce a protein called LAP3 that plants use to make sporopollenin, trying to isolate LAP3 so she could figure out where it falls in the chain of events that leads to sporopollenin production. She and her colleagues in the Weng lab want to understand the mechanism underlying this process because it may give engineers ideas for making strong, flexible, synthetic materials like wearable electronics. Sporopollenin degrades slowly after ingestion, so researchers have also suggested coating drugs with this substance so that they’ll be released gradually once inside the human body.

Elliot is fascinated by this intersection of technology and chemistry, and thinks she might like to center her PhD thesis on a similar topic. In particular, nanotechnology that improves cancer drug delivery has captured her imagination, and she may try to pursue such research at the Koch Institute at MIT after she graduates from Howard University.

Purifying LAP3 was a tricky task, as a portion of the protein that targets it to chloroplasts also made it difficult to separate from the bacteria Elliot was using to produce it. Removing this chloroplast targeting sequence made purifying LAP3 possible, but only in combination with a bacterial protein called a chaperone that is typically responsible for binding other proteins to make sure they maintain functional conformations. Elliot tested the function of LAP3 and the chaperone together, and found that they use a water molecule to break apart another protein in the sporopollenin production pathway. Other Weng lab members will continue to try to isolate LAP3 after Elliot leaves, in order to confirm the activity they observed can truly be attributed to this protein.

As Elliot solidified her knowledge of biochemistry, she formed lasting relationships with the people in her lab and in her MSRP-Bio cohort. Walcott wrote of feeling “burdened” by his conflicting loves for St. Lucia, where he wanted to live, and for writing, which necessitated leaving. When Elliot first moved to the United States, she truly understood these poems for the first time, as her island’s warm, familiar faces and wave-strewn shores were suddenly replaced with an unfamiliar culture and bitter winters. At MIT, Elliot found a fantastic group of coworkers who embodied the St. Lucian spirit of friendship. “Everyone in my lab treated me like I was one of them,” she says. “They reached out to me to strike up conversations, tell me funny stories, and just talk to me.”

Outside of the Weng lab, Elliot’s MSRP-Bio cohort also provided a wealth of friendship. For the first time, she found peers who truly shared her passion for research. The students all lived in an MIT dorm, where their conversations went on long into the night. “We’d go on and on about our experiments,” she says. “It was like a vortex of science.”

While Walcott and Lewis motivated Elliot to aim high, her time at MIT gave her the technical skills to handle whatever challenges science throws at her. “The MSRP program has made me quite savvy about the way research works,” she says. Combined with the St. Lucian spirit of working hard and always striving for success, Elliot is returning to Howard with the full array of qualities that will help her become the “hard working, efficient, and impactful researcher” that she wants to be.

Photo credit: Saima Sidik