Research Area: Biochemistry, Biophysics, and Structural Biology

Directing evolution in search of a better plastic-degrading enzyme

Graduate student En Ze Linda Zhong-Johnson is creating new methods to measure and enhance enzyme activity — which she hopes will help restore a plastic-choked world.

Grace van Deelen
April 21, 2022

After graduating with her undergraduate degree in molecular genetics from the University of Toronto in 2016, En Ze Linda Zhong-Johnson celebrated with a trip to Alaska. There, she saw a pristine landscape unlike the plastic-littered shores of the Toronto waterfront. “What I saw up there was so different from what I saw in the city,” Zhong-Johnson says. “I realized there shouldn’t be all this waste floating everywhere, in our water, in our environment. It’s not natural.”

As a trained biologist, Zhong-Johnson began to think about the problem of plastic pollution from a biological perspective. One solution, she thought, could be biological recycling: a process by which living organisms break down materials, using digestion or other metabolic processes to turn these materials into smaller pieces or new compounds. Composting, for example, is a type of biological recycling — microbes in the soil break down discarded food, speeding up the decomposition process. Zhong-Johnson wondered if there were any organisms on Earth that could use the carbon in polyethylene terephthalate (PET), a common plastic used in water bottle and food packaging, as an energy source.

Earlier that same year, Japanese scientists discovered that a bacterium, Ideonella sakaiensis, could do just that by producing enzymes that could break down PET. The two main PET-degrading enzymes, referred to as IsPETase and IsMHETase, are able to turn PET into two chemical compounds, terephthalic acid and ethylene glycol, which I. sakaiensis can use for food.

The discovery of these enzymes opened up many new questions and possible applications that scientists have continued to work on since. However, because there was — and still is — much to learn about PET-degrading enzymes, they are still not widely used to recycle consumer products. Zhong-Johnson figured that, in graduate school, she could build on the existing IsPETase research and help to accelerate their use at recycling facilities. Specifically, she wanted to engineer the enzyme to work faster at lower temperatures, and study how, fundamentally, the enzymes worked on the surface of PET plastic to degrade it.

“I hopped on the excitement train, along with the rest of the world,” she says.

A better enzyme

After receiving her acceptance to MIT to complete her PhD, Zhong-Johnson approached various professors, pitching her idea to speed up IsPETase activity. Christopher Voigt, the Daniel I. C. Wang Professor of Biological Engineering, and Anthony Sinskey, professor of biology, were interested, and formed a co-advisorship to support Zhong-Johnson’s project. Sinskey, in particular, was impressed by her idea to help solve the world’s plastic problem with PET-degrading enzymes.

Woman pipetting in lab
Zhong-Johnson screens for enzyme variants with improved activity. Credit: Grace van Deelen

“Plastic pollution is a big problem,” he says, “and that’s the kind of problem my lab likes to tackle.” Plus, he says, he feels “committed to helping graduate students who want to apply their science and technology learnings to the environment.”

While the idea of a plastic-degrading enzyme seems like a panacea, the enzyme’s practical applications have been limited by its biology. The wild-type IsPETase is a mesophilic enzyme, meaning the structure of the enzyme is only stable around ambient temperatures, and the enzyme loses its activity above that threshold. This restriction on temperature limits the number and types of facilities that can use IsPETase, as well as the rate of the enzyme reaction, and drives up the cost of their use.

However, Zhong-Johnson thinks that, with combined approaches of biological and chemical engineering, it’s possible to scale up the use of the enzymes by increasing their stability and activity. For example, an enzyme that’s highly active at lower temperatures could work in unheated facilities, or even be sprinkled directly into landfills or oceans to degrade plastic waste — a process called bioremediation. Increasing the activity of the enzyme at ambient temperatures could also expand the possible applications.

“Most of the environments where plastic is present are not above 50 degrees Celsius,” said Zhong-Johnson. “If we can increase enzyme activity at lower temperatures, that’s really interesting for bioremediation purposes.”

Now a fifth-year graduate student, Zhong-Johnson has honed her project, and is focusing on increasing the activity of IsPETase. To do so, she’s using directed evolution — creating random mutations in the IsPETase gene, and selecting for IsPETase variants that digest PET faster. When they do, she combines the beneficial mutations and uses that as template for the next round of library generation, to improve the enzyme even further. The evolution is “directed” because Zhong-Johnson herself, rather than nature, is picking out which gene sequences of enzyme proceed through to the next round of random mutagenesis, and which don’t. Her ultimate goal is to create a more efficient and hardier enzyme that will, hopefully, work faster at ambient temperatures.

A better protocol

Just as Zhong-Johnson was beginning her project, she ran into an obstacle: There wasn’t a standard way to measure whether her experiments were successful. In particular, no immediately applicable method existed to measure enzyme kinetics for IsPETases on solid substrates like plastic bottles and other plasticware. That was a problem for Zhong-Johnson because understanding enzyme activity was a crucial part of how she selected her enzymes in the directed evolution process.

Usually, enzyme activity is measured via product accumulation: When enzymes metabolize a substance, they create a new substance in return, called a product. Measuring the amount of product created by an enzyme after a certain amount of time gives the researcher a snapshot of that enzyme’s activity.

There are two problems with the product accumulation method, though. First, it is usually done using liquid or soluble substrates. In other words, the material that the enzyme is targeting is dissolved, like sugar dissolved in water. Then, the enzyme is added to that liquid concoction and mixed evenly throughout. However, the substrate Zhong-Johnson wanted to use — PET — was not soluble but solid, meaning it could not be evenly distributed like a soluble substrate. Second, the product accumulation measurement methods available were only practical for measuring less than a handful of timepoints for a few enzyme or substrate concentrations. As a result, many in the field opted to measure a single time point, late in the enzyme reaction, which doesn’t provide an indication of how an enzyme’s rate of digestion actually changes over the course of time — something that can be measured through kinetic measurements.

Taking kinetic measurements would help researchers like Zhong-Johnson illustrate the full pattern of enzyme activity and answer questions like: When is the enzyme most active? Does most product accumulation happen at the beginning of the reaction or the end? How does temperature impact the rate of these reactions over time? To answer these questions, she realized she would have to develop the method herself. 

Through a serendipitous discussion with a group of chemical engineering undergraduate students that Zhong-Johnson was mentoring, she came up with a solution, which she published in a 2021 paper in Scientific Reports. The undergraduates brought to her attention many factors that she had overlooked about the enzyme, and she says she would not have realized the importance of kinetic measurements if it weren’t for the fact that she was trying to design an experiment that the undergraduates could perform over the course of three hours.

The paper outlined a new way to measure enzyme activity, which Zhong-Johnson calls “the bulk absorbance method.” Instead of measuring the final product accumulation at very late time points, the bulk absorbance method involves taking multiple kinetic measurements at early time intervals during the experiment. This technique informs Zhong-Johnson’s directed evolution approach: If she can find which enzymes are most active at low temperatures, she can select the best possible enzyme for the next round of analyses. She hasn’t yet engineered an enzyme she’s completely happy with, but she’s gotten much closer to her ultimate goal.

Solving big problems together

Zhong-Johnson’s discoveries have been made possible by the collaboration between her and her two co-advisors, Voigt and Sinskey, who have supported her independence throughout her five years at MIT.

Man and woman smile by whiteboard
Zhong-Johnson and her advisor, professor Anthony Sinskey, in his office. Credit: Grace van Deelen

When she first started her graduate work, neither Voigt nor Sinskey had expertise in enzyme biochemistry involving solid substrates: Sinkey’s lab focuses on bacterial metabolism, while Voigt’s lab focuses on genetic engineering (though Voigt did have experience with directed evolution research). Additionally, Zhong-Johnson’s path to her project was rather unconventional. Most grad students do not come to potential advisors proposing entire dissertations, which posed a unique challenge for Zhong-Johnson.

Despite not having specific expertise in enzyme biochemistry involving solid substrates, Voigt and Sinskey have supported Zhong-Johnson in other ways: by helping her to develop critical thinking skills and connecting her to other people in her field, such as potential collaborators, who can help her project thrive in the future. Zhong-Johnson has supplemented her MIT experience by having enzyme experts as part of her dissertation committee as well.

Sinskey says that, in the future — once Zhong-Johnson has engineered the ideal enzyme — they would like to partner with industry, and work on making the enzyme into a product that waste companies might use to recycle plastic. Additionally, Sinskey says, the plastic problem and the IsPETase solution raise so many interesting questions that Zhong-Johnson’s project will probably live on in the Voigt and Sinskey labs even after she graduates. He’d like to see other graduate students working to understand the enzyme’s activity and progressing the directed evolution that Zhong-Johnson started.

Zhong-Johnson is already working on understanding the specifics of how IsPETase act on PET. “How does it eat a hole in a plastic bottle? How does it move along and make the hole bigger as it moves through the process? Does it jump around? Or does it keep degrading a single polymer chain until its completely broken down? We just don’t know the answers yet,” says Sinskey.

But Zhong-Johnson is up to the task. “My graduate students have to have three skills, in my opinion,” Sinskey says. “One, they have to be intelligent. Two, they have to be energetic, and three, they have to be of high integrity, in research and behavior.” Zhong-Johnson, he says, has all three qualities.

Using plant biology to address climate change

A Climate Grand Challenges flagship project aims to reduce agriculture-driven emissions while making food crop plants heartier and more nutritious.

Merrill Meadow | Whitehead Institute
April 20, 2022

On April 11, MIT announced five multiyear flagship projects in the first-ever Climate Grand Challenges, a new initiative to tackle complex climate problems and deliver breakthrough solutions to the world as quickly as possible. This article is the fourth in a five-part series highlighting the most promising concepts to emerge from the competition and the interdisciplinary research teams behind them.

The impact of our changing climate on agriculture and food security — and how contemporary agriculture contributes to climate change — is at the forefront of MIT’s multidisciplinary project “Revolutionizing agriculture with low-emissions, resilient crops.” The project The project is one of five flagship winners in the Climate Grand Challenges competition, and brings together researchers from the departments of Biology, Biological Engineering, Chemical Engineering, and Civil and Environmental Engineering.

“Our team’s research seeks to address two connected challenges: first, the need to reduce the greenhouse gas emissions produced by agricultural fertilizer; second, the fact that the yields of many current agricultural crops will decrease, due to the effects of climate change on plant metabolism,” says the project’s faculty lead, Christopher Voigt, the Daniel I.C. Wang Professor in MIT’s Department of Biological Engineering. “We are pursuing six interdisciplinary projects that are each key to our overall goal of developing low-emissions methods for fertilizing plants that are bioengineered to be more resilient and productive in a changing climate.”

Whitehead Institute members Mary Gehring and Jing-Ke Weng, plant biologists who are also associate professors in MIT’s Department of Biology, will lead two of those projects.

Promoting crop resilience

For most of human history, climate change occurred gradually, over hundreds or thousands of years. That pace allowed plants to adapt to variations in temperature, precipitation, and atmospheric composition. However, human-driven climate change has occurred much more quickly, and crop plants have suffered: Crop yields are down in many regions, as is seed protein content in cereal crops.

“If we want to ensure an abundant supply of nutritious food for the world, we need to develop fundamental mechanisms for bioengineering a wide variety of crop plants that will be both hearty and nutritious in the face of our changing climate,” says Gehring. In her previous work, she has shown that many aspects of plant reproduction and seed development are controlled by epigenetics — that is, by information outside of the DNA sequence. She has been using that knowledge and the research methods she has developed to identify ways to create varieties of seed-producing plants that are more productive and resilient than current food crops.

But plant biology is complex, and while it is possible to develop plants that integrate robustness-enhancing traits by combining dissimilar parental strains, scientists are still learning how to ensure that the new traits are carried forward from one generation to the next. “Plants that carry the robustness-enhancing traits have ‘hybrid vigor,’ and we believe that the perpetuation of those traits is controlled by epigenetics,” Gehring explains. “Right now, some food crops, like corn, can be engineered to benefit from hybrid vigor, but those traits are not inherited. That’s why farmers growing many of today’s most productive varieties of corn must purchase and plant new batches of seeds each year. Moreover, many important food crops have not yet realized the benefits of hybrid vigor.”

The project Gehring leads, “Developing Clonal Seed Production to Fix Hybrid Vigor,” aims to enable food crop plants to create seeds that are both more robust and genetically identical to the parent — and thereby able to pass beneficial traits from generation to generation.

The process of clonal (or asexual) production of seeds that are genetically identical to the maternal parent is called apomixis. Gehring says, “Because apomixis is present in 400 flowering plant species — about 1 percent of flowering plant species — it is probable that genes and signaling pathways necessary for apomixis are already present within crop plants. Our challenge is to tweak those genes and pathways so that the plant switches reproduction from sexual to asexual.”

The project will leverage the fact that genes and pathways related to autonomous asexual development of the endosperm — a seed’s nutritive tissue — exist in the model plant Arabidopsis thaliana. In previous work on Arabidopsis, Gehring’s lab researched a specific gene that, when misregulated, drives development of an asexual endosperm-like material. “Normally, that seed would not be viable,” she notes. “But we believe that by epigenetic tuning of the expression of additional relevant genes, we will enable the plant to retain that material — and help achieve apomixis.”

If Gehring and her colleagues succeed in creating a gene-expression “formula” for introducing endosperm apomixis into a wide range of crop plants, they will have made a fundamental and important achievement. Such a method could be applied throughout agriculture to create and perpetuate new crop breeds able to withstand their changing environments while requiring less fertilizer and fewer pesticides.

Creating “self-fertilizing” crops

Roughly a quarter of greenhouse gas (GHG) emissions in the United States are a product of agriculture. Fertilizer production and use accounts for one third of those emissions and includes nitrous oxide, which has heat-trapping capacity 298-fold stronger than carbon dioxide, according to a 2018 Frontiers in Plant Science study. Most artificial fertilizer production also consumes huge quantities of natural gas and uses minerals mined from nonrenewable resources. After all that, much of the nitrogen fertilizer becomes runoff that pollutes local waterways. For those reasons, this Climate Grand Challenges flagship project aims to greatly reduce use of human-made fertilizers.

One tantalizing approach is to cultivate cereal crop plants — which account for about 75 percent of global food production — capable of drawing nitrogen from metabolic interactions with bacteria in the soil. Whitehead Institute’s Weng leads an effort to do just that: genetically bioengineer crops such as corn, rice, and wheat to, essentially, create their own fertilizer through a symbiotic relationship with nitrogen-fixing microbes.

“Legumes such as bean and pea plants can form root nodules through which they receive nitrogen from rhizobia bacteria in exchange for carbon,” Weng explains. “This metabolic exchange means that legumes release far less greenhouse gas — and require far less investment of fossil energy — than do cereal crops, which use a huge portion of the artificially produced nitrogen fertilizers employed today.

“Our goal is to develop methods for transferring legumes’ ‘self-fertilizing’ capacity to cereal crops,” Weng says. “If we can, we will revolutionize the sustainability of food production.”

The project — formally entitled “Mimicking legume-rhizobia symbiosis for fertilizer production in cereals” — will be a multistage, five-year effort. It draws on Weng’s extensive studies of metabolic evolution in plants and his identification of molecules involved in formation of the root nodules that permit exchanges between legumes and nitrogen-fixing bacteria. It also leverages his expertise in reconstituting specific signaling and metabolic pathways in plants.

Weng and his colleagues will begin by deciphering the full spectrum of small-molecule signaling processes that occur between legumes and rhizobium bacteria. Then they will genetically engineer an analogous system in nonlegume crop plants. Next, using state-of-the-art metabolomic methods, they will identify which small molecules excreted from legume roots prompt a nitrogen/carbon exchange from rhizobium bacteria. Finally, the researchers will genetically engineer the biosynthesis of those molecules in the roots of nonlegume plants and observe their effect on the rhizobium bacteria surrounding the roots.

While the project is complex and technically challenging, its potential is staggering. “Focusing on corn alone, this could reduce the production and use of nitrogen fertilizer by 160,000 tons,” Weng notes. “And it could halve the related emissions of nitrous oxide gas.”

Probing how proteins pair up inside cells

MIT biologists drilled down into how proteins recognize and bind to one another, informing drug treatments for cancer.

Raleigh McElvery | Department of Biology
February 3, 2022

Despite its minute size, a single cell contains billions of molecules that bustle around and bind to one another, carrying out vital functions. The human genome encodes about 20,000 proteins, most of which interact with partner proteins to mediate upwards of 400,000 distinct interactions. These partners don’t just latch onto one another haphazardly; they only bind to very specific companions that they must recognize inside the crowded cell. If they create the wrong pairings — or even the right pairings at the wrong place or wrong time — cancer or other diseases can ensue. Scientists are hard at work investigating these protein-protein relationships, in order to understand how they work, and potentially create drugs that disrupt or mimic them to treat disease.

The average human protein is composed of approximately 400 building blocks called amino acids, which are strung together and folded into a complex 3D structure. Within this long string of building blocks, some proteins contain stretches of four to six amino acids called short linear motifs (SLiMs), which mediate protein-protein interactions. Despite their simplicity and small size, SLiMs and their binding partners facilitate key cellular processes. However, it’s been historically difficult to devise experiments to probe how SLiMs recognize their specific binding partners.

To address this problem, a group led by Theresa Hwang PhD ’21 designed a screening method to understand how SLiMs selectively bind to certain proteins, and even distinguish between those with similar structures. Using the detailed information they gleaned from studying these interactions, the researchers created their own synthetic molecule capable of binding extremely tightly to a protein called ENAH, which is implicated in cancer metastasis. The team shared their findings in a pair of eLife studies, one published on Dec. 2, 2021, and the other published Jan. 25.

“The ability to test hundreds of thousands of potential SLiMs for binding provides a powerful tool to explore why proteins prefer specific SLiM partners over others,” says Amy Keating, professor of biology and biological engineering and the senior author on both studies. “As we gain an understanding of the tricks that a protein uses to select its partners, we can apply these in protein design to make our own binders to modulate protein function for research or therapeutic purposes.”

Most existing screens for SLiMs simply select for short, tight binders, while neglecting SLiMs that don’t grip their partner proteins quite as strongly. To survey SLiMs with a wide range of binding affinities, Keating, Hwang, and their colleagues developed their own screen called MassTitr.

The researchers also suspected that the amino acids on either side of the SLiM’s core four-to-six amino acid sequence might play an underappreciated role in binding. To test their theory, they used MassTitr to screen the human proteome in longer chunks comprised of 36 amino acids, in order to see which “extended” SLiMs would associate with the protein ENAH.

ENAH, sometimes referred to as Mena, helps cells to move. This ability to migrate is critical for healthy cells, but cancer cells can co-opt it to spread. Scientists have found that reducing the amount of ENAH decreases the cancer cell’s ability to invade other tissues — suggesting that formulating drugs to disrupt this protein and its interactions could treat cancer.

Thanks to MassTitr, the team identified 33 SLiM-containing proteins that bound to ENAH — 19 of which are potentially novel binding partners. They also discovered three distinct patterns of amino acids flanking core SLiM sequences that helped the SLiMs bind even tighter to ENAH. Of these extended SLiMs, one found in a protein called PCARE bound to ENAH with the highest known affinity of any SLiM to date.

Next, the researchers combined a computer program called dTERMen with X-ray crystallography in order understand how and why PCARE binds to ENAH over ENAH’s two nearly identical sister proteins (VASP and EVL). Hwang and her colleagues saw that the amino acids flanking PCARE’s core SliM caused ENAH to change shape slightly when the two made contact, allowing the binding sites to latch onto one another. VASP and EVL, by contrast, could not undergo this structural change, so the PCARE SliM did not bind to either of them as tightly.

Inspired by this unique interaction, Hwang designed her own protein that bound to ENAH with unprecedented affinity and specificity. “It was exciting that we were able to come up with such a specific binder,” she says. “This work lays the foundation for designing synthetic molecules with the potential to disrupt protein-protein interactions that cause disease — or to help scientists learn more about ENAH and other SLiM-binding proteins.”

Ylva Ivarsson, a professor of biochemistry at Uppsala University who was not involved with the study, says that understanding how proteins find their binding partners is a question of fundamental importance to cell function and regulation. The two eLife studies, she explains, show that extended SLiMs play an underappreciated role in determining the affinity and specificity of these binding interactions.

“The studies shed light on the idea that context matters, and provide a screening strategy for a variety of context-dependent binding interactions,” she says. “Hwang and co-authors have created valuable tools for dissecting the cellular function of proteins and their binding partners. Their approach could even inspire ENAH-specific inhibitors for therapeutic purposes.”

Hwang’s biggest takeaway from the project is that things are not always as they seem: even short, simple protein segments can play complex roles in the cell. As she puts it: “We should really appreciate SLiMs more.”

Cellular environments shape molecular architecture

Researchers glean a more complete picture of a complex structure called the nuclear pore complex by studying it directly inside cells.

Raleigh McElvery | Department of Biology
October 13, 2021

Context matters. It’s true for many facets of life, including the tiny molecular machines that perform vital functions inside our cells.

Scientists often purify cellular components, such as proteins or organelles, in order to examine them individually. However, a new study published today in the journal Nature suggests that this practice can drastically alter the components in question.

The researchers devised a method to study a large, donut-shaped structure called the nuclear pore complex (NPC) directly inside cells. Their results revealed that the pore had larger dimensions than previously thought, emphasizing the importance of analyzing complex molecules in their native environments.

“We’ve shown that the cellular environment has a significant impact on large structures like the NPC, which was something we weren’t expecting when we started,” says Thomas Schwartz, the Boris Magasanik Professor of Biology at MIT and the study’s co-senior author. “Scientists have generally thought that large molecules are stable enough to maintain their fundamental properties both inside and outside a cell, but our findings turn that assumption on its head.”

In eukaryotes like humans and animals, most of a cell’s DNA is stored in a rounded structure called the nucleus. This organelle is shielded by the nuclear envelope, a protective barrier that separates the genetic material in the nucleus from the thick fluid filling the rest of the cell. But molecules still need a way to come in and out of the nucleus in order to facilitate important processes, including gene expression. That’s where the NPC comes in. Hundreds — sometimes thousands — of these pores are embedded in the nuclear envelope, creating gateways that allow certain molecules to pass.

The study’s first author, former MIT postdoc Anthony Schuller, compares NPCs to gates at a sports stadium. “If you want to access the game inside, you have to show your ticket and go through one of these gates,” he explains.

The NPC may be tiny by human standards, but it’s one of the largest structures in the cell. It’s comprised of roughly 500 proteins, which has made its structure challenging to parse. Traditionally, scientists have broken it up into individual components to study it piecemeal using a method called X-ray crystallography. According to Schwartz, the technology required to analyze the NPC in a more natural environment has only recently become available.

Together with researchers from the University of Zurich, Schuller and Schwartz employed two cutting-edge approaches to solve the pore’s structure: cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET).

An entire cell is too thick to look at under an electron microscope. But the researchers sliced frozen colon cells into thin layers using the cryo-FIB equipment housed at MIT.nano’s Center for Automated Cryogenic Electron Microscopy and the Koch Institute for Integrative Cancer Research’s Peterson (1957) Nanotechnology Materials Core Facility. In doing so, the team captured cross-sections of the cells that included NPCs, rather than simply looking at the NPCs in isolation.

“The amazing thing about this approach is that we’ve barely manipulated the cell at all,” Schwartz says. “We haven’t perturbed the cell’s internal structure. That’s the revolution.”

What the researchers saw when they looked at their microscopy images was quite different from existing descriptions of the NPC. They were surprised to find that the innermost ring structure, which forms the pore’s central channel, is much wider than previously thought. When it’s left in its natural environment, the pore opens up to 57 nanometers — resulting in a 75 percent increase in volume compared to previous estimates. The team was also able to take a closer look at how the NPC’s various components work together to define the pore’s dimensions and overall architecture.

“We’ve shown that the cellular environment impacts NPC structure, but now we have to figure out how and why,” Schuller says. Not all proteins can be purified, he adds, so the combination of cryo-ET and cryo-FIB will also be useful for examining a variety of other cellular components. “This dual approach unlocks everything.”

“The paper nicely illustrates how technical advances, in this case cryo-electron tomography on cryo-focused ion beam milled human cells, provide a fresh picture of cellular structures,” says Wolfram Antonin, a professor of biochemistry at RWTH Aachen University in Germany who was not involved in the study. The fact that the diameter of the NPC’s central transport channel is larger than previously thought hints that the pore could have impressive structural flexibility. “That may be important for the cell to adapt to increased transport demands,” Antonin explains.

Next, Schuller and Schwartz hope to understand how the size of the pore affects which molecules can pass through. For instance, scientists only recently determined that the pore was big enough to allow intact viruses like HIV into the nucleus. The same principle applies to medical treatments: only appropriately-sized drugs with specific properties will be able to access the cell’s DNA.

Schwartz is especially curious to know whether all NPCs are created equal, or if their structure differs between species or cell types.

“We’ve always manipulated cells and taken the individual components out of their native context,” he says. “Now we know this method may have much bigger consequences than we thought.”

Rewiring cell division to make eggs and sperm
Whitehead Institute
July 30, 2021

To create eggs and sperm, cells must rewire the process of cell division. Mitosis, the common type of cell division that our bodies use to grow everything from organs to fingernails and to replace aging cells, produces two daughter cells with the same number of chromosomes and approximately the same DNA sequence as the original cell. Meiosis, the specialized cell division that makes egg and sperm in two rounds of cell division, creates four granddaughter cells with new variations in their DNA sequence and half as many chromosomes in each cell. Meiosis uses most of the same cellular machinery as mitosis to achieve this very different outcome; only a few key molecular players prompt the rewiring from one type of division to another. One such key player is the protein Meikin, which is found exclusively in cells undergoing meiosis.

New research from Whitehead Institute Member Iain Cheeseman, graduate student Nolan Maier and collaborators Professor Michael Lampson and senior research scientist Jun Ma at the University of Pennsylvania demonstrates how Meikin is elegantly controlled, and sheds light on how the protein acts to serve multiple roles over different stages of meiosis. The findings, which appear in Developmental Cell on July 30, reveal that Meikin is precisely cut in half midway through meiosis. Instead of this destroying the protein, one half of the molecule, known as C-Meikin, goes on to play a critical role as a previously hidden protein actor in meiosis.

“Cells have this fundamental process, mitosis, during which they have to divide chromosomes evenly or it will cause serious problems like cancer, so the system has to be very robust,” Maier says. “What’s incredible is that you can add one or two unique meiotic proteins like Meikin and dramatically change the whole system very quickly.”

Helping chromosomes stick together

During both mitosis and meiosis, sister chromatids — copies of the same chromosome — pair up to form the familiar “X” shape that we recognize as a chromosome. In mitosis, each chromatid—each half of the X — is connected to a sort of cellular fishing line and these lines reel the chromatids to opposite ends of the cell, where the two new cells are formed around them. However, in the first round of division in meiosis, the sister chromatids stick together, and one whole “X” is reeled into each new cell. Meikin helps to achieve this different outcome by ensuring that, while the chromosomes are being unstuck from each other in preparation for being pulled apart, each pair of sister chromatids stays glued together in the right place. Meikin also helps ensure that certain cellular machinery on the sister chromatids is fused so that they will connect to the same line and be reeled together to the same side of the cell.

More specifically, when chromosomes are first paired up, they are glued together by adhesive molecules in three regions: the centromere, or center of the X, where Meikin localizes; the region around the center; and the arms of the X. In the first round of meiosis, Meikin helps to keep the glue in the region around the center intact, so the sister chromatids will stick together. Simultaneously, Meikin helps to prime the center region to be unglued, while a separate process unglues the arms. This ungluing allows the chromosomes to separate and be prepared for later stages of meiosis.

Cheeseman and Maier initially predicted that Meikin’s role ended after meiosis I, the first round of meiotic cell division. In meiosis II, the second round of cell division, the cells being created should end up with only one sister chromatid each, and so the chromatids must not be kept glued together. Maier found that near the end of meiosis I, Meikin is cleaved in two by an enzyme called Separase, the same molecule that cleaves the adhesive molecules gluing together the chromosomes. At first, this cleavage seemed like the end of Meikin and the end of this story.
A hidden role for a hidden proteinHowever, unexpectedly, the researchers found that cells lacking Meikin during the second half of meiosis do not divide properly, prompting them to take another look at what happens to Meikin after it gets cleaved. They found that Separase cleaves Meikin at a specific point — carving it with the precision of a surgeon’s scalpel — to create C-Meikin, a previously unknown protein that turns out to be necessary for meiosis II. C-Meikin has many of the same properties as the intact Meikin molecule, but it is just different enough to take on a different role: helping to make sure that the chromosomes align properly before their final division.

“There’s a lot of protein diversity in cells that you would never see if you don’t go looking for it, if you only look at the DNA or RNA. In this case, Separase is creating a completely different protein variant of Meikin than can function differently in meiosis II,” says Cheeseman, who is also a professor of biology at Massachusetts Institute of Technology. “I’m very excited to see what we might discover about other hidden protein forms in cell division.”

Recombining ideas

Answering the question of Meikin’s role and regulation throughout meiosis required a close collaboration and partnership between Maier and Lampson lab researcher Ma – the Lampson lab being experts on studying meiosis using mouse models. Working with mouse oocytes (immature egg cells), Ma was able to reveal the behaviors and critical contributions of Meikin cleavage in meiotic cells in mice. Both labs credit the close exchange with helping them to get a deeper understanding of how cells rewire for meiosis.

“It was a pleasure working together to understand how some of the specialized meiotic functions that are necessary for making healthy eggs and sperm are controlled,” Lampson says.

Finally, once cells have completed these specialized meiotic divisions, the researchers found that it was critical for oocytes to fully eliminate Meikin. The researchers determined that, after meiosis two, C-Meikin is degraded by another molecule (the anaphase-promoting complex or APC/C)—this time for good. With Meikin gone and the rewiring of cell division reversed, eggs and sperm are ready for mitosis; should they fuse and form an embryo, that is the next cell division they will undergo. The researchers note that the way Meikin is regulated by being broken down — first into C-Meikin and then completely — may help cells to organize their timing during meiosis. Breaking apart a protein is an irreversible step that creates a clear demarcation between before and after in a multi-step process.The researchers hope that by uncovering the intricacies of meiosis, they may shed light on what happens when the creation of eggs and sperm goes wrong, and so perhaps contribute to our understanding of infertility. Cheeseman also hopes that by studying how mitotic processes are rewired for meiosis, his lab can gain new insights into the original wiring of mitosis.

Yadira Soto-Feliciano

Education

  • PhD, 2016, MIT
  • BS, 2008, Chemistry, University of Puerto Rico-Mayagüez 

Research Summary

We study chromatin — the complex of DNA and proteins that make up our chromosomes. We aim to understand how post-translational modifications to these building-blocks, as well as the factors that regulate these events, play essential roles in maintaining the integrity of cells, tissues, and ultimately entire organisms. We implement a combination of functional genomics, biochemical, genetic, and epigenomic approaches to study how chromatin and epigenetic factors decode the chemical language of chromatin, and how these are dysregulated in diseases such as cancer.

Awards

  • AACR Gertrude B. Elion Cancer Research Award, 2023
  • V Foundation Award, 2022
  • NIH MOSAIC K99/R00 Postdoctoral Career Transition Award, 2021
  • Eddie Méndez Scholar Award, Fred Hutchinson Cancer Research Center, 2020
  • Damon Runyon-Sohn Pediatric Cancer Fellowship, Damon Runyon Cancer Research Foundation, 2017
Two heads are better than one, but two disciplines are even better

How biologists and mathematicians reached across departmental lines to solve a long-standing problem in electron microscopy

Saima Sidik | Department of Biology
April 19, 2021

MIT’s Hockfield Court is bordered on the west by the ultra-modern Stata Center, with its reflective, silver alcoves that jut off at odd angles, and on the east by Building 68, which is a simple, window-lined, cement rectangle. At first glance, Bonnie Berger’s mathematics lab in the Stata Center and Joey Davis’s biology lab in Building 68 are as different as the buildings that house them. And yet, a recent collaboration between these two labs shows how their disciplines complement each other. The partnership started when Ellen Zhong, a graduate student from the Computational and Systems Biology (CSB) Program, decided to use a computational pattern-recognition tool called a neural network to study the shapes of molecular machines. Three years later, Zhong’s project is letting scientists see patterns that run beneath the surface of their data, and deepening their understanding of the molecules that shape life.

Zhong’s work builds on a technique from the 1970s called cryo-electron microscopy (cryo-EM), which lets researchers take high-resolution images of frozen protein complexes. Over the last decade, better microscopes and cameras have led to a “resolution revolution” in cryo-EM that’s allowed scientists to see individual atoms within proteins. But, as good as these images are, they’re still only static snapshots. In reality, many of these molecular machines are constantly changing shape and composition as cells carry out their normal functions and adjust to new situations.

Along with former Berger lab member Tristan Belper, Zhong devised software called cryoDRGN. The tool uses neural nets to combine hundreds of thousands of cryo-EM images, and shows scientists the full range of three-dimensional conformations that protein complexes can take, letting them reconstruct the proteins’ motion as they carry out cellular functions. Understanding the range of shapes that protein complexes can take helps scientists develop drugs that block viruses from entering cells, study how pests kill crops, and even design custom proteins that can cure disease. COVID-19 vaccines, for example, work partly because they include a mutated version of the virus’s spike protein that’s stuck in its active conformation, so vaccinated people produce antibodies that block the virus from entering human cells. Scientists needed to understand the variety of shapes that spike proteins can take in order to figure out how to force spike into its active conformation.

Two women standing by rock wall
Graduate student Ellen Zhong (right), and her co-advisor, Professor of Mathematics Bonnie Berger (left)

Getting off the computer and into the lab

Zhong’s interest in computational biology goes back to 2011 when, as a chemical engineering undergrad at the University of Virginia, she worked with Professor Michael Shirts to simulate how proteins fold and unfold. After college, Zhong took her skills to a company called D. E. Shaw Research, where, as a Scientific Programmer, she took a computational approach to studying how proteins interact with small molecule drugs.

“The research was very exciting,” Zhong says, “but all based on computer simulations. To really understand biological systems, you need to do experiments.”

This goal of combining computation with experimentation motivated Zhong to join MIT’s CSB PhD program, where students often work with multiple supervisors to blend computational work with bench work. Zhong “rotated” in both the Davis and Berger labs, then decided to combine the Davis lab’s goal of understanding how protein complexes form with the Berger lab’s expertise in machine learning and algorithms. Davis was interested in building up the computational side of his lab, so he welcomed the opportunity to co-supervise a student with Berger, who has a long history of collaborating with biologists.

Davis himself holds a dual bachelor’s degree in computer science and biological engineering, so he’s long believed in the power of combining complementary disciplines. “There are a lot of things you can learn about biology by looking in a microscope,” he says. “But as we start to ask more complicated questions about entire systems, we’re going to require computation to manage the high-dimensional data that come back.”

Before rotating in the Davis lab, Zhong had never performed bench work before — or even touched a pipette. She was fascinated to find how streamlined some very powerful molecular biology techniques can be. Still, Zhong realized that physical limitations mean that biology is much slower when it’s done at the bench instead of on a computer. “With computational research, you can automate experiments and run them super quickly, whereas in the wet lab, you only have two hands, so you can only do one experiment at a time,” she says.

Zhong says that synergizing the two different cultures of the Davis and Berger labs is helping her become a well-rounded, adaptable scientist. Working around experimentalists in the Davis lab has shown her how much labor goes into experimental results, and also helped her to understand the hurdles that scientists face at the bench. In the Berger lab, she enjoys having coworkers who understand the challenges of computer programming.

“The key challenge in collaborating across disciplines is understanding each other’s ‘languages’,” Berger says. “Students like Ellen are fortunate to be learning both biology and computing dialects simultaneously.”

Bringing in the community

Man smiling outside
Zhong’s second co-advisor, Professor Joey Davis

Last spring revealed another reason for biologists to learn computational skills: these tools can be used anywhere there’s a computer and an internet connection. When the COVID-19 pandemic hit, Zhong’s colleagues in the Davis lab had to wind down their bench work for a few months, and many of them filled their time at home by using cryo-EM data that’s freely available online to help Zhong test her cryoDRGN software. The difficulty of understanding another discipline’s language quickly became apparent, and Zhong spent a lot of time teaching her colleagues to be programmers. Seeing the problems that non-programmers ran into when they used cryoDRGN was very informative, Zhong says, and helped her create a more user-friendly interface.

Although the paper announcing cryoDRGN was only recently published, the tool created a stir as soon as Zhong posted her code online, many months prior. The cryoDRGN team thinks this is because leveraging knowledge from two disciplines let them visualize the full range of structures that protein complexes can have, and that’s something researchers have wanted to do for a long time. For example, the cryoDRGN team recently collaborated with researchers from Harvard and Washington University to study locomotion of the single-celled organism Chlamydomonas reinhardtii. The mechanisms they uncovered could shed light on human health conditions, like male infertility, that arise when cells lose the ability to move. The team is also using cryoDRGN to study the structure of the SARS-CoV-2 spike protein, which could help scientists design treatments and vaccines to fight coronaviruses.

Zhong, Berger, and Davis say they’re excited to continue using neural nets to improve cryo-EM analysis, and to extend their computational work to other aspects of biology. Davis cited mass spectrometry as “a ripe area to apply computation.” This technique can complement cryo-EM by showing researchers the identities of proteins, how many of them are bound together, and how cells have modified them.

“Collaborations between disciplines are the future,” Berger says. “Researchers focused on a single discipline can take it only so far with existing techniques. Shining a different lens on the problem is how advances can be made.”

Zhong says it’s not a bad way to spend a PhD, either. Asked what she’d say to incoming graduate students considering interdisciplinary projects, she says: “definitely do it.”

Spying on enzymes while they perform chemical reactions could help treat gut ailments
Raleigh McElvery
March 26, 2021

Humans breathe oxygen, but many microbes deep within in our gut don’t have access to this precious resource. Instead, they breathe sulfur compounds, releasing hydrogen sulfide in the process. This colorless gas is best-known for its rotten stench, but inside the human colon it has been linked to a thinner mucus barrier, and ailments such as inflammatory bowel disease, Crohn’s disease, ulcerative colitis, and colorectal cancer. In order to develop potential treatments, researchers are probing how microbes create hydrogen sulfide and which molecules they use.

To help further these efforts, Catherine Drennan’s lab and Heather Kulik’s lab at MIT collaborated with Emily Balskus’ lab at Harvard University to investigate the structure and mechanism of an enzyme that’s critical for hydrogen sulfide production: isethionate sulfite-lyase (IslA). The team examined IslA while it was bound to a metabolite that’s readily available in the gut — and revealed how the bacterium Bilophila wadsworthia uses this interaction to help generate the hydrogen sulfide precursor called sulfite. The researchers then compared IslA’s binding behavior to other enzymes in the same family, in order to better understand how these enzymes have evolved to perform challenging chemistry on a wide variety of molecules. Their findings were published on Mar. 26 in the journal Cell Chemical Biology.

“Although abundant, sulfide-producing bacteria are not well understood,” says Drennan, a professor of biology and chemistry and a Howard Hughes Medical Institute investigator. “By characterizing the enzymes in these bacteria that are responsible for sulfur metabolism, we can develop therapeutic strategies to limit production of hydrogen sulfide that can lead to disease.”

Although researchers have been studying bacterial sulfur respiration for decades, IslA was only recently identified. This enzyme breaks the bond between a carbon atom and a sulfur atom in a compound called isethionate, which is a prevalent metabolite in the human body. In doing so, IslA releases the sulfite that bacteria such as B. wadsworthia use to produce hydrogen sulfide.

IslA is a member of a large family of enzymes, known as glycyl radical enzyme (GREs). Scientists can learn a lot from examining the way GREs bind to other molecules, according to Christopher Dawson PhD ’20, the study’s co-first author.

GREs contain a binding site (or “active site”) where they latch onto their respective substrates to perform chemical reactions. “Understanding GREs better will aid in drug design efforts to combat the deleterious effects of some of these enzymes,” Dawson says. “It will also help to engineer enzymes that perform diverse, challenging reactions to expand the toolkit for chemical synthesis.”

To this end, Dawson wanted to compare IslA’s active site — where it binds to isethionate to break the C-S bond — to other enzymes in the GRE family. He used X-ray crystallography to visualize this interaction at the level of individual atoms. The GREs he examined shared similar “barrel-like” structures in their active sites, but used these core features in different ways, depending on the substrates they bound. For instance, isethionate bound higher in IslA’s active site compared to the way other GREs bind their respective substrates. While this aberrant binding behavior is quite unique — even among GREs — another group had found something similar when they elucidated IslA’s structure in a different bacterium. And, the Drennan lab suspects this pattern could be prevalent in other classes of enzymes as well.

Next, Dawson and his colleagues wanted to investigate how IslA goes about cleaving the C-S bond once the enzyme has bound to isethionate. Others had predicted this process would occur via a “migration” reaction. In that scenario, the sulfite leaving group first migrates to another carbon atom and then that C-S bond is cleaved to release it. However, after co-first author Stephania Irwin generated multiple IslA variants, the Kulik lab performed computational analyses, and the researchers completed structural comparisons, the team concluded that migration was not occurring. Instead, IslA appeared to be performing an “elimination” reaction that severed the C-S bond without forming another one via migration.

Now that they know more about IslA — and GREs in general — the researchers hope their insights will aid drug design.

“Understanding how pathogens use enzymes like these to extract sulfite from their hosts and fuel hydrogen sulfide production has very clear therapeutic implications,” Dawson says. “And that’s one of the things I like best about this story.”

Citation
“Molecular Basis of C-S Bond Cleavage in the Glycyl Radical Enzyme Isethionate Sulfite-Lyase”
Cell Chemical Biology, online March 26, 2021,
DOI: 10.1016/j.chembiol.2021.03.001
Christopher D. Dawson, Stephania M. Irwin, Lindsey R. F. Backman, Chip Le, Jennifer X. Wang, Vyshnavi Vennelakanti, Zhongyue Yang, Heather J. Kulik, Catherine L. Drennan, and Emily P. Balskus

Members of MIT Biology came together with alumni, industry representatives, and supporters to review the department’s challenges and accomplishments.

March 9, 2021

Seychelle Vos investigates how the genome is organized so it can fit inside the cell — and how that careful organization affects gene expression.

February 24, 2021