Research Area: Biochemistry, Biophysics, and Structural Biology

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

Undergraduate Meucci Ilunga spent 10 weeks investigating protein interactions, exploring career options, and making new friends.

Saima Sidik

September 4, 2019

Meucci Ilunga seems to know something about everything. He’s a videographer who’s branching out into podcasting. He’s researched cancer therapies and volunteered in a hospital. He grew up on a Navajo reservation, and he’s a year away from completing a biochemistry degree at the University of Arizona. “I’m excited about life in general,” he says. At the moment, though, he’s especially excited about a cellular conundrum that he investigated during the 10-week internship in the MIT Department of Biology that he completed as part of the MIT Summer Research Program in Biology (MSRP-Bio).

“Your cells are really, really complicated,” he says. “They’re packed with lots of different kinds of proteins. Yet when you look at how proteins interact, they’re specific.” How do proteins find the appropriate binding partners amongst all the noise? Ilunga and his MSRP-Bio supervisor, biology and biological engineering Professor Amy Keating, think that short sequences of amino acids — the units that comprise proteins — can mediate binding interactions more intricate than researchers had previously appreciated.

Just as proteins home in on their binding partners, Ilunga has always been drawn to science. As a kid, he told everyone he wanted to be an astrophysicist. “I had no idea what that meant,” he says, “but I loved the idea of exploring the unknown and being able to generate knowledge.”

Ilunga grew up on the Navajo reservation in Kinlichee, Arizona, however, and he didn’t have the same opportunities to engage in science as kids in urban centers. “Only about 60 percent of people on the reservation have running water and electricity,” he says, “so most people are pressed with more urgent matters than following their curiosities.”

Ilunga notes the myriad of difficulties his reservation faces, from prevalent diabetes to corrupt politicians and poor school systems, but says that the hardest part about being Navajo is feeling like his people’s problems are invisible to those outside the tribe. “A lot of us feel very forgotten about,” he says.

Ilunga quickly exhausted the opportunities that his high school in Fort Defiance, Arizona, had to offer, leading him to graduate early and leave for the University of Arizona at age 16. But he was determined to remember his roots. Balancing his love of science with his connection to the reservation — and finding a career that will let him return — has proven challenging.

“You can become an engineer, but there are no engineering jobs on the reservation. You can become a computer scientist, but there are no computer science jobs,” he says. So he decided to pursue biochemistry, as it would lay the foundation for medical school, and the reservation is always in need of doctors.

At his university, Ilunga started shadowing physicians and volunteering in a hospital. His path to medical school seemed clear. There was only one problem: He found medicine unfulfilling. “There’s so much more I could be doing. So I started looking at what else I could do to get back home,” he says.

This desire for balance is what made Ilunga choose to join the MSRP-Bio program, for which he received sponsorship from the Gould Fund. Ilunga met the MSRP-Bio coordinator, Mandana Sassanfar, at a conference for minority students, and she told him that MSRP-Bio promotes a balance between lab work and life. “What sold me on this program is that it understands that I’m more than just a scientist,” he says.

Over the summer, Ilunga has spoken with many MIT professors about the diverse professional paths scientists can take, and these conversations have inspired him to consider a career in policy.

“I could be someone who goes to Congress to fight — not only for Native American affairs, but also for scientific affairs,” he says.

Ilunga plans to pursue a PhD in life sciences in preparation for this career, possibly studying protein interactions like the ones he’s been working on all summer. He finds research most interesting when it has a clear clinical application, and understanding protein interactions lets researchers design drugs that disrupt them.

The protein interactions that Ilunga researched are mediated by sequences called short linear motifs, or SLiMs, which consist of contiguous stretches of only three to 10 amino acids — a small subset of the hundreds of amino acids that make up the typical protein. While larger domains are able to form tighter and more sustained interactions, SLiMs mediate weaker, transient interactions.

SLiMs make up in speed what they lack in strength. Allowing proteins to quickly bind and release each other is beneficial for some biological processes, and SLiMs can also evolve rapidly and let organisms adapt to change quickly. Researchers think this is why SLiMs have persisted in many different organisms over the course of evolution, despite being relatively unintuitive tools for forming protein complexes. The Keating lab noticed that sometimes proteins that contain SLiMs recognize their binding partners with a specificity that’s unexpected, given that so many proteins contain these short sequences.

Ilunga spent his summer looking into how small domains and short sequences can play a large role in protein pairing. His weeks began with culturing large quantities of bacteria that were used to produce SLiM-containing peptides; then he isolated these peptides and used a technique called biolayer interferometry to determine how tweaking their amino acid sequences affected how strongly they bound their target protein.

When he altered the amino acid sequence directly adjacent to the SLiMs, Ilunga found that the strength of their binding interactions could vary quite wildly. The Keating lab doesn’t understand how this occurs, and Ilunga’s findings pave the way for testing different biochemical mechanisms to explain this phenomenon.

When he wasn’t isolating proteins or chatting with the MIT faculty, Ilunga got to know the MIT community. “At a lot of top schools there’s a sense of prestige that fills the air, but it wasn’t like that at MIT. Everyone here is so humble,” he says.

He especially enjoyed getting to know his fellow MSRP-Bio students. Whether they were going on a boat cruise along the Charles River or helping each other troubleshoot lab work, he says it was an amazing group of people to spend the summer with.

As he heads back to the University of Arizona, Ilunga is taking many technical skills back with him, as well as a new outlook on life. He has always been hopeful that life will get easier for Navajos and other minorities. Now he’s confident that the medical and technological advances that institutions like MIT are creating can improve living conditions for people like his family back on the reservation.

“I used to think my optimism was blind,” he says. “Now I think my optimism is informed.”

Graduate student Rohan Jonnalagadda analyzes the 3D shapes of iron-containing enzymes to parse their role in cellular processes.

Raleigh McElvery

August 27, 2019

Raised in a computer-savvy family well-versed in software and information technology, Rohan Jonnalagadda had a strong desire to “decode” the world around him. But his kind of code, the genetic one, consists of four repeating letters: A, T, C, and G. “Just like a computer runs on software, I wanted to investigate the code behind the molecular hardware that gives rise to life,” he says. Now a sixth-year graduate student in the Drennan lab, he works to decrypt the structure of metal-containing proteins, in order to determine the roles they play in vital cellular reactions.

When Jonnalagadda was an undergraduate biochemistry major at the University of California, Berkeley, it became clear to him that the genetic code was more than just a string of letters; it also serves as the blueprint for all the proteins in the entire organism. These proteins fold into complex 3D structures, which ultimately beget function.

At UC Berkeley, he joined a lab studying the iron-containing protein Heme-Nitric Oxide/Oxygen (H-NOX) that senses nitric oxide gas in bacterial and eukaryotic cells. When H-NOX binds to nitric oxide, it must change its 3D shape in the process. Jonnalagadda used a technique known as X-ray crystallography to freeze H-NOX in various stages of this conformational change to determine how it binds the gas molecules.

“I think we sometimes ignore the fact that we need trace metals in order to survive,” he says. “I was interested in continuing to think about what different metals could do in the cell. And using metals opens up a whole new world of chemical reactions that you generally don’t learn about in class.”By the time he graduated and began his PhD at MIT Biology, Jonnalagadda had been using X-ray crystallography for over two years. Today, as a member of Catherine Drennan’s lab, he continues to leverage this same method to parse the structure of additional metal-containing proteins.

In fact, the two projects that he’s devoted most of his time to over the past five years involve reactions that he’d never even heard of before he arrived at MIT. The focus of his first undertaking was the iron-containing enzyme ribonucleotide reductase (RNR), which helps generate deoxyribonucleotides, the building blocks of DNA.

Jonnalagadda aims to understand how this enzyme is regulated to ensure the cell maintains the proper amount of each type of deoxyribonucleotide, in order to properly replicate and repair its genome. If those ratios are incorrect, the cell could experience detrimental stress.

Because the enzyme is regulated differently in humans than it is in bacteria, scientists hope to one day create antibiotics that target the bacterial RNR while leaving the human RNR unscathed. Jonnalagadda works with the human version, devising an assay that will allow him to better assess the differences between the two enzymes. RNR is notoriously difficult to work with, and so Jonnalagadda has spent much of his time developing ways to purify it so it remains stable.

His second project is a collaboration with researchers at his alma mater, UC Berkeley, investigating isonitriles — compounds containing a carbon atom tripled bonded to a nitrogen atom. Because isonitriles are used to make drugs like antibiotics, scientists have a keen interest in exploring new ways to produce them. The team discovered that one bacterium, Streptomyces coeruleorubidus, had a novel and mysterious way of synthesizing these compounds. Jonnalagadda wants to know exactly how these particular bacteria do it.

He is using X-ray crystallography to determine the structure of the iron-containing enzyme ScoE in S. coeruleorubidus, which is responsible for forming the carbon-nitrogen triple bond characteristic of isonitriles.

“It’s exciting to be working on a protein that’s only just been discovered,” he says. “There’s just so much more to learn about its fundamental biological function. I think that’s why basic research is so appealing to me; you never know where the work will take you, or the impacts it could have on human health later on.”

Extending the frontiers of any discipline requires some guesswork and metaphorical bushwhacking, and Jonnalagadda has learned almost as much from his failed experiments as he has from his successful ones. “I’m proud that I’ve been able to use what I’ve learned about experimental design to help others in my lab when they have questions,” he says.

As he considers life post-graduation, he hopes to use the biochemical and structural techniques he’s mastered over the years to secure a job in industry.

“Being part of a department with such broad and wide-ranging research interests has made it easy to see that my work doesn’t exist in a vacuum,” he says. “It connects to many different aspects of biology.”

Photo credit: Raleigh McElvery
Posted 8.23.19

Saima Sidik

July 17, 2019

Millennia ago, before the evolution of multicellular life, bacteria developed a group of proteins called glycyl radical enzymes to help them turn food into cellular energy. Glycyl radical enzymes functioned efficiently for millions of years until bacteria encountered a new hurdle: oxygen build-up in the atmosphere. Glycyl radicals are easily damaged by oxygen, and bacteria needed a way to continue to process nutrients under these new conditions.

In a recent study published in Journal of Biological Inorganic Chemistry, Sarah Bowman et al. from MIT provide structural evidence for how bacteria use “spare part” proteins to repair glycyl radical enzymes by replacing their damaged portions.

“Instead of degrading the whole protein and building a new one, bacteria make a small spare part protein that can bind to the glycyl radical enzyme and restore its function,” says Lindsey Backman, a graduate student in the MIT Department of Chemistry and co-author on the study.

Senior author Catherine Drennan, an MIT professor of biology and chemistry and a Howard Hughes Medical Institute Investigator, is leading the effort to characterize a glycyl radical enzyme called pyruvate formate lyase, or PFL, and its corresponding spare part protein, YfiD. Using a technique called nuclear magnetic resonance spectroscopy, her lab examined the shape of YfiD. Previous work revealed that upon oxygen exposure, part of PFL is cleaved off, leaving the enzyme unable to perform its chemistry. Then, using in silico modeling, the Drennan lab showed that YfiD fits neatly into the hole in oxygen-damaged PFL, replacing the missing piece.

“If you have a car and you get a flat tire, you don’t buy a new car, you just change the tire,” Backman says.

Although YfiD is one of only two spare part proteins that have been identified, Drennan suspects there are many more. It’s easy for researchers to mistake spare parts for the portions of proteins that they replace, and Drennan thinks that scientists have probably overlooked spare parts when analyzing the complex milieu of proteins found in cells.

Although identifying spare parts is technically challenging, Drennan thinks the applications for synthetic biology make it well worth the effort. Specifically, she’s interested in bacteria that live in the deep sea where there’s very little oxygen and use other glycyl radical enzymes to degrade hydrocarbons. Because they can break down hydrocarbons, these bacteria could be valuable tools for cleaning up oil spills. Oil spills occur on the ocean’s surface where there’s a lot of oxygen, so spare parts might be necessary to stabilize oil-degrading proteins under these conditions.

“The question always becomes, ‘what about the oxygen sensitivity?’” Drennan says, referring to these oil-degrading proteins. “What if we expressed a spare part to make them more stable?”

Backman began working on spare part proteins as an undergraduate participating in the MIT Summer Research Program, or MSRP, as did several of the study’s other authors, and Drennan says that this project’s success highlights the valuable role these students play in MIT labs. Now a full-time graduate student, Backman has teamed up with Drennan lab postdoc and co-author Mary Andorfer, and together they plan to continue characterizing the interaction between YfiD and PFL. They think their findings may be the first step in showing that spare parts are a common protein repair mechanism, and that characterizing them will add a new tool to the synthetic biology toolbox.

Citation:
“Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme”
JBIC Journal of Biological Inorganic Chemistry, online June 26, 2019, DOI: 10.1007/s00775-019-01681-2.
Sarah E. J. Bowman, Lindsey R. F. Backman, Rebekah E. Bjork, Mary C. Andorfer, Santiago Yori, Alessio Caruso, Collin M. Stultz, and Catherine L. Drennan.