Research Area: Biochemistry, Biophysics, and Structural Biology
Greta Friar | Whitehead Institute
May 5, 2020
What do the painkillers morphine and codeine, the cancer chemotherapy drug vinblastine, the popular brain health supplement salidroside, and a plethora of other important medicines have in common? They are all produced in plants through processes that rely on the same family of enzymes, the aromatic amino acid decarboxylases (AAADs). Plants, which have limited ability to physically react to their environments, have instead evolved to produce a stunning array of chemicals that allow them to do things like deter pests, attract pollinators, and adapt to changing environmental conditions. A lot of these molecules have also turned out to be useful in medicine—but it’s unusual for one family of enzymes to be responsible for so many different molecules of importance to both plants and humans. New research from Whitehead Institute Member Jing-Ke Weng, who is also an associate professor of biology at the Massachusetts Institute of Technology, and postdoctoral researcher Michael Torrens-Spence delves into the science behind the AAADs’ unusual generative capacity.
Plants create their useful molecules through biochemical pathways made up of chains of enzymes. Each enzyme acts as an assembly worker, taking in a molecule—starting with a basic building block like an amino acid—and performing biochemical modifications in sequence. The altered molecules get passed down the line until the last enzyme creates the final natural product. Once the pathway enzymes for a molecule of interest have been identified, researchers can copy their corresponding genes into organisms like yeast and bacteria that are capable of producing the molecules at scale more easily than the original plants. The AAAD family of enzymes function as gatekeepers to plants’ specialized molecule production because they operate at the beginning of many of the enzyme assembly lines; they take various amino acids, molecules that are widely available in nature, and direct them into different enzymatic pathways that produce unique molecules that only exist in plants. When an AAAD evolves to perform a new function, as has occurred frequently in their evolutionary history, this change high up in the assembly lines can cascade into the development of new biochemical pathways that create new natural products—leading to the diversity of medicines that stem from AAAD-gated pathways.
Due to the AAADs’ prominent role in the production of medically important molecules, Weng and Torrens-Spence decided to investigate how the AAADs came to be so prolific. In research published in the journal PNAS on May 5, the researchers illuminate the structural and functional underpinnings of the AAADs’ diversity. They also demonstrate how their detailed knowledge of the enzymes can be used to engineer novel enzymatic pathways to produce important molecules of interest from plants.
“We characterized these enzymes very thoroughly, which is a great starting place for manipulating the system and engineering it to do something new. That’s particularly exciting when you’re dealing with enzymes at the interface between primary and specialized plant metabolism; it can apply to a lot of downstream drugs,” Torrens-Spence says.
The AAAD family evolved from one ancestral enzyme into a diverse set of related enzymes over a relatively short period of time. This sort of diversification occurs when an enzyme gets accidentally duplicated, after which one copy has evolutionary pressure on it to maintain the same function, but the other copy suddenly has free range to evolve. If the superfluous enzyme mutates to do something new that is useful to the organism, from then on both enzymes, with their distinct roles, are likely to be maintained. In the case of the AAADs, this process occurred many times, leading to a large number of enzymes that appear almost exactly alike, yet can do very different things.
In order to explain the AAADs’ successful rate of diversification, the researchers took a close look at four enzymes in the AAAD family with different roles, and discovered the composition and three-dimensional shape—the crystal structure—of each. The crystal structure allowed the researchers to see how these molecular machines hold and modify specific molecules; this meant that they could understand why some AAADs initiate certain specialized-molecule production lines while other AAADs initiate alternative production lines. The researchers next used genetics and biochemistry to pinpoint the differences between the enzymes and how small genetic variations enact very major changes to the enzyme’s underlying machinery. This detailed analysis explained, among others things, how a subset of enzymes that evolved out of the AAADs, the aromatic acetaldehyde synthases (AASs), came to perform a completely different action on molecules while still being so similar to true AAADs that the two types of enzymes are often mistaken for each other.
After the researchers developed this thorough understanding of the AAAD family of enzymes, as well as knowledge of the AAAD-containing pathways that create useful medicinal molecules, they applied this knowledge by engineering an entirely new pathway to create a molecule of interest, (S)-norcoclaurine, a precursor molecule for morphine and other poppy-based painkillers. Torrens-Spence combined enzymes from pathways in different species to invent a novel chain of enzyme reactions that can produce (S)-norcoclaurine in fewer steps than is seen in nature. This experiment was a proof of concept that Torrens-Spence says shows the potential for such biosynthetic engineering, for example as a method to produce plant-based drugs more easily.
“Often with these molecules of interest, you figure out the pathway in plants and copy-paste it into a more scalable system, like yeast, that will produce larger quantities of the molecule,” Torrens-Spence says. “Here we’re applying engineering principles to biology, so that we can innovate and build something new.”
Written by Greta Friar
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Jing-Ke Weng’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an associate professor of biology at Massachusetts Institute of Technology.
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Citation:
“Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins”
PNAS, May 5, 2020
DOI: https://doi.org/10.1073/pnas.1920097117
Michael P. Torrens-Spence (1), Ying-Chih Chiang (2†), Tyler Smith (1,3), Maria A. Vicent (1,4), Yi Wang (2), and Jing-Ke Weng (1,3)
1 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
2 Department of Physics, the Chinese University of Hong Kong, Shatin, N.T., Hong Kong.
3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
4 Department of Biology, Williams College, Williamstown, Massachusetts 01267, USA.
† Present address: School of Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
Eva Frederick | Whitehead Institute
April 20, 2020
In overgrown areas from Canada to China, a lush, woody vine with crescent-shaped seeds holds the secret to making a cancer-fighting chemical. Now, Whitehead Institute researchers in Member Jing-Ke Weng’s lab have discovered how the plants do it.
Plants in the family Menispermaceae, from the Greek words “mene” meaning “crescent moon,” and “sperma,” or seed, have been used in the past for a variety of medicinal purposes. Native Americans used the plants to treat skin diseases, and would ingest them as a laxative. Moonseed was also used as an ingredient in curare, a muscle relaxant used on the tips of poison arrows.
But the plants also may have a use in modern-day medicine: a compound called acutumine shown to have anti-cancer properties (although not tested specifically against cancer cells, the chemical has been shown to kill human T-cells, an important quality for leukemia and lymphoma treatments). Acutumine is a halogenated product, which means the molecule is capped on one end by a halogen atom — a group that includes fluorine, chlorine and iodine, among others. In this case, the halogen is chlorine.
Halogenated compounds like acutumine can be useful in medicinal chemistry — their unusual chemical appendages mean they react in interesting ways with other biomolecules, and drug designers can put them to use in creating compounds to complete specific tasks in the body. Today, 20% of pharmaceutical compounds are halogenated. “However, chemists’ ability to efficiently install halogen atoms to desirable positions of starting compounds has been quite limited,” Weng says.
Most natural halogenated products come from microorganisms such as algae or bacteria, and acutumine is one of the only halogenated products made by plants. Chemists finally succeeded in synthesizing the compound in 2009, although the reaction is time-consuming and expensive (10 mg of synthesized acutumine can cost around $2,000).
Colin Kim, a graduate student in the Weng lab at Whitehead Institute, wanted to know how these plants were completing this tricky reaction using only their own genetic material. “We thought, why don’t we ask how the plants make it and then upscale the reaction [to produce it more efficiently]?” Kim says.
“By understanding how living organisms such as the moonseed plant perform chemically challenging halogenation chemistry, we could devise new biochemical approaches to produce novel halogenated compounds for drug discovery,” Weng says.
Kim knew that for every halogenated molecule in an organism, there is an enzyme called a halogenase that catalyzes the reaction that sticks on that halogen. Halogenases are useful in creating pharmaceuticals – a well-placed halogen can help fine-tune the bioactivities of various drugs. So Weng, who is also an associate professor of biology at Massachusetts Institute of Technology, and Kim, who spearheaded the project, began working to identify the helper molecule responsible for creating acutumine in moonseed plants.
First, the scientists obtained three species of Menispermaceae plants. Two of them, common moonseed (Menispermum canadense) and Chinese moonseed (Sinomenium acutum), were known to produce acutumine. They also procured one plant in the same family called snake vine (Stephania japonica) which did not produce the compound.
They began their investigation by using mass spectrometry to look for acutumine in all three plants, and then find out exactly where in the plants it was located. They found the chemical all throughout the first two — and some extra in the roots of common moonseed. As expected, the third plant, snake vine, had none, and could therefore be used as a reference species, since presumably it would not ever express the gene for the halogenase enzyme that could stick on the chlorine molecule.
Next, the researchers started searching for the gene. They began by sequencing the RNA that was being expressed in the plants (RNA serves as a messenger between genomic DNA and functional proteins), and created a huge database of RNA sorted by what tissue it had been identified in.
At this point, the extra acutumine in the roots of common moonseed came in handy. The researchers had some idea of what the enzyme might look like – past research on other halogenases in bacteria suggested that one specific family of enzyme, called Fe(II)/2-oxoglutarate-dependent halogenases, or 2ODHs, for short, was capable of site-specifically adding a halogen in the same way that the moonseed’s mystery enzyme did. Although no 2ODHs had yet been found in plants, the researchers thought this lead was worth a look. So they searched specifically for transcripts similar to 2ODH sequences that were more highly expressed in the roots of common moonseed than in the leaves and stems.
After analyzing the RNA transcripts, Kim and Weng were pretty sure they had found what they were looking for: one gene in particular (which they named McDAH, short for M. canadense dechloroacutumine halogenase) was highly expressed in the roots of common moonseed. Then, in Chinese moonseed, they identified another protein that shared 99.1 percent of McDAH’s sequence, called SaDAH. No similar protein was found in snakevine, suggesting that this protein was likely the enzyme they wanted.
To be sure, the researchers tested the enzyme in the lab, and found that it was indeed the first-ever plant 2ODH, able to stick on the chlorine molecule to the alkaloid molecule dechloroacutumine to form acutumine. Interestingly, the enzyme was pretty picky; when they gave it other alkaloids like codeine and berberine to see if it would install a halogen on those as well, the enzyme ignored them, suggesting it was highly specific toward its preferred substrate, dechloroacutumine, the precursor of acutumine. They compared the enzyme’s activity to other similar enzymes, and found the key to its ability lay in the substitution of one specific amino acid in the active site– aspartic acid — for a glycine.
Now that they had identified the enzyme responsible for the moonseed’s halogenation reactions, Kim and Weng wanted to see what else it could do. A chemical capable of catalyzing such a complex reaction might be useful for chemists trying to synthesize other compounds, they hypothesized.
So they presented the enzyme with some dechloroacutumine and a whole buffet of alternative anions to see whether it might catalyze a reaction with any of these molecules in lieu of chlorine. Of the selection of anions, including bromide, azide, and nitrogen dioxide, the enzyme catalyzed a reaction only with azide, a construct of 3 nitrogen atoms.
“That is super cool, because there isn’t any other naturally occurring azidating enzyme that we know of,” Kim says. The enzyme could be used in click chemistry, a nature-inspired method to create a desired product through a series of simple, easy reactions.
In future studies, Weng and Kim hope to use what they’ve learned about the McDAH and SaDAH enzymes as a starting point to create enzymes that can be used as tools in drug development. They’re also interested in using the enzyme on other plant products to see what happens. “Plant natural products, even without chlorines, are pretty effective and bioactive, so it would be cool to see if you can take those plant natural products and then install chlorines to see what kind of changes and bioactivity it has, whether it develops new-to-nature functions or retain its original bioactivity with enhanced properties,” Kim says. “It expands the biocatalytic toolbox we have for natural product biosynthesis and its derivatization.”
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Written by Eva Frederick
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Citation: Kim, Colin Y. et al. The chloroalkaloid (−)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants. Nature Communications. April 20, 2020. DOI: 10.1038/s41467-020-15777-w
March 30, 2020
National Institute of Environmental Health Sciences
March 2, 2020
Graham Walker, Ph.D., studies the processes cells use to repair and tolerate DNA damage from environmental pollutants. For more than 40 years, he has worked to understand how cells respond to DNA damage, and how these processes can introduce mutations that lead to cancer and other human diseases.
His current NIEHS-funded work focuses on translesion synthesis (TLS). This damage tolerance process allows specialized enzymes that copy DNA, called TLS DNA polymerases, to replicate past lesions in damaged DNA. The process can help cells tolerate environmental DNA damage, but because TLS polymerases frequently insert the wrong DNA base, they can also lead to DNA mutations.
“The TLS process is critically important to human health because it helps cells survive DNA damage, but it can come at a cost,” said Walker. “It isn’t the kind of repair system you would think we would want because it makes a lot of mistakes. However, as we drill into these details, we are finding that there is so much more to be learned than just the strict biochemistry.”
In 2017, Walker was one of eight environmental health scientists to receive an inaugural Revolutionizing Innovative, Visionary Environmental Health Research (RIVER) Outstanding Investigator Award from NIEHS. The grant, which funds researchers rather than specific projects, provides Walker with flexibility to explore novel directions in his research.
From the Ames Test to TLS
Walker was drawn into the world of DNA repair and mutagenesis as a postdoctoral fellow at the University of California, Berkeley, under the guidance of Bruce Ames, Ph.D. Ames’ group created the Ames test, still used today, to determine whether a given chemical is likely to cause cancer. The Ames test uses bacterial strains that include a derivative of a naturally occurring drug-resistant plasmid, a small circular DNA molecule, known as pKM101. This molecule significantly increases the mutation rate of bacterial genes in response to chemical exposures, playing an important role in this quick and convenient test to estimate carcinogenic potential.
“I decided there must be something really interesting on that plasmid because it led to much higher mutation rates in bacteria for the same amount of damage,” said Walker.
After arriving at the Massachusetts Institute of Technology, his current employer, Walker continued to study the mechanisms behind these mutations.
Walker and his research team discovered the specific genes of pKM101 that are needed for it to produce more mutations. They showed that these genes are orthologs, or genes that evolved from a common ancestral gene, in the Escherichia coli (E. coli) chromosome that are required for the bacteria to mutate in response to DNA damage. This work helped lay the groundwork for the discovery of TLS DNA polymerases and how they are controlled.
“When we first sequenced these genes, nothing like them had been previously reported, but subsequently more and more related genes were discovered in all domains of life,” said Walker. “After decades of work by many labs, we now know that these are all TLS DNA polymerases and that the pKM101 plasmid encodes a polymerase that is responsible for the increased mutations.”
Using Bacteria to Understand DNA Damage
Walker’s prior research on the mutagenesis-enhancing function of pKM101 also led him to analyze E. coli’s SOS system, a set of biological responses that are activated to rescue cells from severe DNA damage. Walker and his team identified genes turned on by DNA damage that are regulated as part of E. coli’s SOS response. Many of the genes encode functions involved in DNA repair or mutagenesis. This work on the SOS response of E. coli was the first to directly demonstrate, in any organism, that DNA damage from environmental sources can change gene expression.
By further exploring TLS DNA polymerases in E. coli, he also identified the biological role of one of the most conserved DNA-damage response enzymes, DinB, which encodes a TLS DNA polymerase, and reported that the gene is required for resistance to some DNA-damaging agents. His work on DinB also suggested an additional mechanism by which antibiotics can become toxic to bacterial cells.
Blocking TLS in Cancer
“While a postdoc in the mid 1970’s with Bruce Ames, my ambitious hope was that by studying pKM101, I would learn something about the fundamental mechanism of how mutations arose in bacteria and humans, and might even learn how to control it,” said Walker. “That is now happening with my current, NIEHS-funded work.”
Some tumors can withstand damage from chemotherapy drugs by relying on TLS, which allows them to survive by replicating past damaged DNA caused by the drugs. In eukaryotes, including humans, mutagenic TLS is carried by two TLS DNA polymerases known as Rev1 and Pol zeta.
In addition to his innovative research, Walker is devoted to improving education and helping undergraduate students. In 2002, Walker became a Howard Hughes Medical Institute Professor and used his funding to establish a science education group modeled on his laboratory research group.
“I feel that training the next generations of scientists is as important as the science itself, and I have been incredibly lucky to have a spectacular set of grad students and post docs work with me over the years,” said Walker. “I have tried to focus as much on training, through teaching and mentoring, as on advancing the science.”
“Not only are these TLS polymerases responsible for introducing a lot of mutations that cause cancer, they also help cancer cells survive in the face of chemotherapy drugs that introduce DNA damage that would otherwise kill them,” said Walker.
Recently, Walker and his colleagues discovered that a small molecule and compound known as JH-RE-06 can block the Rev1-Pol zeta mutagenic TLS pathway by interfering with the ability of the Rev1 domain to recruit Pol zeta. The researchers tested the molecule in human cancer cell lines and showed that it enhanced the ability of several different types of chemotherapy to kill cancer cells, while also suppressing their ability to mutate in the presence of DNA-damaging drugs. In a mouse model of human melanoma, they found that not only did the tumors stop growing in mice treated with a combination of the chemotherapy drug cisplatin and JH-RE-06, those mice also survived longer.
“I am able to take more chances and try more high-risk experiments with the RIVER award,” said Walker. “The flexibility and extra resources are now allowing me to identify TLS inhibitors, which are offering startlingly unexpected mechanistic insights and also show potential to improve chemotherapy.”
A change in fields and a two-body problem ultimately led Biology and BE Professor Amy Keating to MIT to study coiled-coils and other protein interactions.
J. Carota | CSB Grad Office
December 17, 2019
About 330 miles west of Cambridge lies the small academic town of Ithaca, New York: the location of Cornell University and the hometown of Professor Amy Keating. Surrounded by academics (her father is a professor of computer science at Cornell), Keating was eager to continue her education after high school—just not in Ithaca.
“I could have stayed at Cornell, which is obviously an extremely good school in my hometown, but my family and I agreed that it was important that I go away,” recounts Keating. The scholar/athlete set her sights on Harvard University based on the excellent rowing team and outstanding academics. Physics particularly appealed to her, because it involved using math to explain mechanical and electrical phenomena, and she chose this as her major. She likes to tell people that she also “attempted pure math but failed miserably.” Keating admits that she was not very good at the abstract subject material, and tackling it side-by-side with math whizzes was a harsh awakening after performing well throughout high school. She switched to studying applied math, which was easier for her to manage and also more useful for a physics major.
With an intense rowing schedule, Keating often found herself working late into the night, struggling to solve problems alone. It took a year or two and a serious injury for her to realize that that most of the physics majors were working together in the library many afternoons while she was on the river. “That was very eye-opening. Now I’m a strong advocate of students teaching each other and learning from each other,” explains Keating.
Graduate study gridlock
As she approached the end of her senior year, she had no doubt that she would pursue a PhD, but she did face a crisis about what to study. Initially, she thought she would go to graduate school for physics and applied to and visited many schools. However, she was troubled by the fact that she had tried out a number of areas of physics but never found one that truly captured her interest. In addition to this, Keating began dating a young man, now her husband, who was majoring in chemistry and not set to graduate for another year. “I learned a lot of organic chemistry from him and got very interested in the subject.”
With the decision made to stay in Cambridge for an additional year, she picked up part time work at a Harvard student residence hall cooking, baking, and cleaning in exchange for room and board. Keating also took a few chemistry courses for credit, coached adult rowing, and spent the rest of her time working in the lab of Harvard Physics Professor Mara Prentiss. By the end of that year, she had developed a keen interest in the field of computational chemistry. Having faced difficult decisions about her own post-college plans, she has “a lot of empathy for students who are twenty-one and trying to decide what they want to do in the world.”
Keating and her future husband applied to the same chemistry PhD program at UCLA, where they were both admitted and joined separate labs. She looks back at the interview weekend at UCLA and remembers one faculty interviewer who pointed out the lack of chemistry in her background. “We were talking about cooking, and I told him I like to cook and had been cooking for a job. He said ‘if you can cook, you can do chemistry’, and there is some truth to that, of course.” Keating acknowledges that the first few months of graduate school were traumatic. “I had exactly two undergrad chemistry classes under my belt. I didn’t really know much chemistry and then I was thrown into this PhD program with chemistry majors. And I was taking graduate level courses with my husband, who is a brilliant chemist. But I caught up and managed to learn a lot in a short time.”
Graduate life smoothed out when Keating joined the lab of Ken Houk, a leader in computational physical organic chemistry. Later in her doctoral studies, she added co-advisor Miguel Garcia-Garibay, an expert in experimental photochemistry. Having the two advisors worked out well and led to several joint publications over Keating’s graduate school career. After her husband’s advisor left UCLA for a company, the couple “had to decide what to do. So, we decided we should graduate quickly.” Now married, Keating and her husband earned their PhDs in under five years, but they would continue to be challenged by the “two-body problem” as they formulated a plan for after graduation.
Further afield
The couple knew they both wanted to find postdoc positions, so they looked in cities like San Diego, San Francisco, and Boston, where positions were abundant. Of that time, Keating says: “I was thinking about different problems or fields where my background might apply. I was reading a lot, just to find out what was out there.” This also marks the first time that she started thinking about problems in biology. “I was actually interested in two areas: material science, and biochemistry, both of which are exciting and rapidly growing areas where chemical principles are centrally important.” Keating’s hard work landed her a position back in Cambridge, where she was again co-advised, this time by former MIT Biology Professor Peter Kim at the Whitehead Institute and MIT Professor of Chemistry Bruce Tidor(who was later the founding director of the CSB PhD Program).
The postdoc transition was another time in Keating’s life that she good-naturedly describes as “traumatic,” as she once again had to work to understand all-new vocabulary and experimental methods. Her postdoc provided Keating with her first exposure to large molecules; it was also when she first started working on protein interactions, which would become the crux of her future research. It was in the Kim Lab that she was introduced to coiled-coil proteins. With her background in physics and chemistry, the simplified repeating interactions in these molecules appealed to her. A principle the Keating Lab continues to follow to this day is that they try not to study the most complicated interactions in biology, but rather simpler interactions that they seek to understand in fine detail.
More two-body problems
After four years, Keating hit the academic job market, but she wasn’t sure if she would be accepted as a biochemist because of her change in fields as a postdoc . Her concerns were short-lived as she ended up with a number of exciting offers, including one from MIT. Keating’s husband decided he would go into industry in Boston and with this decision she accepted MIT’s offer to join the Biology faculty in 2002. Later, she added a joint appointment in Biological Engineering.
Keating offers advice to students who are dealing with the two-body problem as she once did.“I think something that helped me and my husband is that we stayed in sync. So, we never had one person make a decision without knowing how that would impact the options of the other person. Of course, that’s not possible for everybody. But that did make our trajectory easier. We would collect our options, put them on the table, look for overlap, and then try to figure out what decision would work best for both of us. And we were very fortunate that we had good options. People have to be flexible to make this work out.” She also recommends looking in cities where there is a high density of opportunities.
The general interest of the Keating lab is in protein-protein interactions, how they work in nature, and how they can be re-engineered using computational and experimental methods. Her group studies proteins that regulate critical processes but are also relatively simple. For example, a system the Keating lab is attracted to is the Bcl-2 family of proteins that control cell death. They have developed a variety of methods that can be used to reprogram the interaction between proteins, and applying these methods to Bcl-2 proteins has generated short peptide molecules that inhibit processes that keep cancer cells alive. Recently the lab has been investigating other types of interactions in cells that are structurally different from the Bcl-2 family. Switching protein families challenges them to develop new methods and allows them to continue to change and evolve their research.
Students and postdocs from the Keating lab have gone on a wide variety of jobs where they study proteins and their interactions in both academia and industry. Keating is happy that young scientists today have so many options. She reflects: “When I was finishing my postdoc, the range of jobs in industry was nothing like it is today. It has been fun to watch my trainees apply their skills to antibody engineering, cancer biology, immuno-oncology and even to start their own companies.” She marvels at how many paths are open to young biologists and likes to tell them that they can’t possibly forsee where they will end up, given the myriad exciting possibilities. Certainly, as a young rower and physics student at Harvard, she had no idea she would end up as a Professor of Biology at MIT.
Biologists devise an efficient method to prepare fluorescently tagged proteins and simulate their native environment.
Raleigh McElvery | Department of Biology
December 18, 2019
All cells have a lipid membrane that encircles their internal components — forming a protective barrier to control what gets in and what stays out. The proteins embedded in these membranes are essential for life; they help facilitate nutrient transport, energy conversion and storage, and cellular communication. They are also important in human disease, and represent around 60 percent of approved drug targets. In order to study these membrane proteins outside the complexity of the cell, researchers must use detergent to strip away the membrane and extract them. However, determining the best detergent for each protein can involve extensive trial and error. And, removing a protein from its natural environment risks destabilizing the folded structure and disrupting function.
In a study published on Dec. 9 in Cell Chemical Biology, scientists from MIT devised a rapid and generalizable way to extract, purify, and label membrane proteins for imaging without any detergent at all — bringing along a portion of the surrounding membrane to protect the protein and simulate its natural environment. Their approach combines well-established chemical and biochemical techniques in a new way, efficiently isolating the protein so it can be fluorescently labeled and examined under a microscope.
“I always joke that it’s not very lifelike to study proteins in soap,” says senior author Barbara Imperiali, a professor of biology and chemistry. “We’ve created a workflow that allows membrane proteins to be imaged while maintaining their native identities and interactions. Hopefully now fewer people will shy away from studying membrane proteins, given their importance in many physiological processes.”
As a member of the Imperiali lab, former postdoc and lead author Jean-Marie Swiecicki investigated membrane proteins from the foodborne pathogen Campylobacter jejuni. In this study, Swiecicki focused on PglC and PglA, two membrane proteins that play a role in enabling the bacteria to infect human cells. His experiments required labeling PglC and PglA with fluorescent tags in order to track them. However, he wasn’t satisfied with existing methods to do so.
In some cases, the fluorescent tags that must be incorporated into the protein in order to visualize it are too large to be placed at defined positions. In other cases, these tags don’t shine brightly enough, or interfere with the structure and function of the protein.
To avoid such issues, Swiecicki decided to use a method known as “unnatural amino-acid mutagenesis.” Amino acids are the units that compose the protein, and unnatural amino-acid mutagenesis involves adding a new amino acid containing an engineered chemical group within the protein sequence. This chemical group can then be labeled with a brightly glowing tag.
Swiecicki inserted the genetic code for the C. jejuni membrane proteins into a different bacterium, Escherichia coli. Inside E. coli, he could incorporate the unnatural amino acid, which could be chemically modified to add the fluorescent label.
When it came time to remove the proteins from the membrane, he substituted a different substance for the detergent: a polymer of styrene-maleic acid (SMA). Unlike detergent, SMA wraps the extracted protein and a small segment of the associated membrane in a protective shell, preserving its native environment. Imperiali explains, “It’s like a scarf protecting your neck from the cold.”
Swiecicki could then monitor the glowing proteins under a microscope to verify his technique was selective enough to isolate individual membrane proteins. The entire process, he says, takes just a few days, and is generally much faster and more reliable than detergent-based extraction methods, which can take months and require the expertise of highly-trained biochemists to optimize.
“I wouldn’t say it’s a magic bullet that’s going to work for every single protein,” he says. “But it’s a highly efficient tool that could make it easier to study many different kinds of membrane proteins.” Eventually, he says, it may even help facilitate high throughput drug screens.
“As someone who works on membrane protein complexes, I can attest to the great need for better methods to study them,” says Suzanne Walker, a professor of microbiology at Harvard Medical School who was not involved in the study. She hopes to extend the approach outlined in the paper to the protein complexes she investigates in her own lab. “I appreciated the extensive detail included in the text about how to apply the strategy successfully,” she adds.
The next steps will be testing the technique on mammalian proteins, and isolating multiple proteins at once in the SMA shell to observe their interactions. And, of course, every new technique deserves a name. “We’re still working on a catchy acronym,” Imperiali says. “Any ideas?”
This research was funded by the Jane Coffin Childs Memorial Fund for Medical Research, Philippe Foundation, and National Institutes of Health.
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
Assistant professors Pulin Li and Seychelle Vos are investigating how cells become tissues and the proteins that organize DNA.
Raleigh McElvery | Department of Biology
September 12, 2019
MIT’s Department of Biology welcomed two new assistant professors in recent months: Pulin Li began at the Whitehead Institute in May, and Seychelle Vos arrived at Building 68 in September. Their respective expertise in genetic circuits and genome organization will augment the department’s efforts to explore cell biology at all levels — from intricate molecular structures to the basis for human disease.
“Pulin and Seychelle bring new perspectives and exciting ideas to our research community,” says Alan Grossman, department head. “I’m excited to see them start their independent research programs and look forward to the impact that they will have.”
From cells to tissues
Growing up in Yingkou, China, Li was exposed to science at a young age. Her dad worked for a pharmaceutical company researching traditional Chinese medicine, and Li would spend hours playing with his lab tools and beakers. “I can still vividly remember the smell of his Chinese herbs,” she says. “Maybe that’s part of the reason why I’ve always been interested in biology as it relates to medical sciences.”
She earned her BS in life sciences from Peking University, and went on to pursue a PhD in chemical biology at Harvard University studying hematopoietic stem cells. Li performed chemical screens to find drugs that would make stem cell transplantation in animal models more efficient, and eventually help patients with leukemia. In doing so, she became captivated by the molecular mechanisms that control cell-to-cell communication.
“I would like to eventually go back to developing new therapies and medicines,” she says, “but that translational research requires a basic understanding of how things work at a molecular level.”
As a result, her postdoc at Caltech was firmly rooted in basic biology. She investigated the genetic circuits that underlie cell-cell communication in developing and regenerating tissues, and now aims to develop new methods to study these same processes here at MIT.
Traditional genetic approaches involve breaking components of a system one at a time to investigate the role they play. However, Li’s lab will adopt a “bottom-up” approach that involves building these systems from the ground up, adding the components back into the cell one by one to pinpoint which genetic circuits are sufficient for programming tissue function. “Building up a system, rather than tearing it down, allows you to test different circuit designs, tune important parameters, and understand why a circuit has evolved to perform a specific function,” she explains.
She is most interested in determining which aspects of cellular communication are critical for tissue formation, in hopes of understanding the diversity of life forms in nature, as well as inspiring new methods to engineer or regenerate different tissues.
“My dream would be to put a bunch of genetic circuits into cells in such a way that they could enable the cells to self-organize into certain patterns and shapes, and replace damaged tissues in a patient,” she says.
Proteins that organize DNA
Although Vos was born in South Africa, her family moved so frequently for her father’s job that she doesn’t call any one place home. “If I had to pick, I’d say it would be the middle of the Atlantic Ocean,” she says.
Both of her grandparents on her mother’s side were researchers, and encouraged various scientific escapades, like bringing wolf spiders to kindergarten for show-and-tell. Her grandmother on her father’s side found her early passions “mildly disturbing,” but dutifully fulfilled her requests for high-resolution insect microscopy books nonetheless.
“I really wanted to know how plants and animals worked starting from a young age, thanks to my grandparents,” Vos says.
In high school she was already conducting research on the side at Clemson University, South Carolina, and went on to earn her BS in genetics from the University of Georgia. She began her PhD in molecular cell biology at the University of California at Berkeley intending to study immunology, but surprised herself by becoming taken with structural biology instead.
Purifying proteins and solving structures required a much different skill set than performing screens and manipulating genomes, but she very much enjoyed her work on topoisomerase, the enzyme that modifies DNA so it doesn’t become too coiled.
She continued conducting biochemical and structural research during her postdoc at the Max Planck Institute for Biophysical Chemistry in Germany. There, she used cryogenic electron microscopy to probe how different RNA polymerase II complexes are regulated during transcription in eukaryotes.
Today, she’s a molecular biologist at her core, but she’s prepared to use “whatever technique gets the answer.” As she explains: “You need biochemistry to solve structures and genetics to understand how they’re working within the whole organism, so it’s all related.”
In her new lab in Building 68, she will continue investigating gene expression, but this time in the context of genome organization. DNA must be compacted in order to fit into a cell, and Vos will study the proteins that organize DNA so it can be compressed without interfering with gene expression. She also wants to know how those same proteins are affected by gene expression.
“How gene regulation impacts compaction is a really critical question to address because different cell types are organized in different ways, and that impacts which genes are ultimately expressed,” she says. “We still don’t really understand how these processes work at an atomic level, so that’s where my expertise in biochemistry and structural biology can be useful.”
When asked what they are most excited about as the school year begins, both Li and Vos say the same thing: the diverse skills and expertise of the students and faculty.
“It’s not just about solving one structure, people here want to understand the entire process,” Vos says. “Biology is a conglomeration of many different fields, and if we can have engineers, mathematicians, physicists, chemists, biologists, and others work together, we can begin to tackle pressing questions.”
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.