New research explores intertwined structures of protein involved in the transfer of biologically essential iron and sulfur clusters
Research from the Drennan Lab in the Department of Biology at MIT explores how a protein called Met18, which is part of a ubiquitous pathway to transfer clusters of iron and sulfur to client proteins in the cytosol and nucleus of cells, can interact with other Met18 units to form intertwined structures
By Lillian Eden
Throughout the cytosol and the nucleus, the delivery of tightly bound sulfur and iron clusters is essential for cellular processes, including DNA replication and repair, ribosome biogenesis, and regulation of proteins. Various enzymes, like helicases, also require these clusters to function. When clusters, called cofactors, aren’t delivered correctly, the client proteins cannot function properly; defects in the cytosolic iron-sulfur cluster assembly (CIA) pathway can lead to human disease.
The molecular machinery responsible for transferring these clusters, called the CIA targeting complex, is not the only molecular machinery responsible for conveying cofactors; other known examples often have only one or two clients. By contrast, the CIA targeting complex can transfer cofactors to more than 30 known clients.
It remains unclear how the CIA targeting complex can be both specific—only transferring iron-sulfur clusters when and where they’re needed—but able to recognize such a wide variety of targets.
New research published today in Communications Biology from the Drennan Lab at MIT in the Department of Biology, Department of Chemistry, and the Howard Hughes Medical Institute focused on the structure and function of a protein called Met18, which may play a role in identifying clients in the CIA pathway. The researchers showed that Met18 can interact with other units of Met18 to form large, intertwined structures, which is perhaps key to how it’s stored when not in use. The work was conducted in collaboration with Melissa D. Marquez and co-workers in the Perlstein Lab in the Department of Chemistry at Boston University.
Previous genetic and biochemical studies indicated Met18 was a key component in the iron-sulfur transfer machinery, but its structure was unknown at the time. Because of the importance of the CIA pathway and the essential nature of iron-sulfur clusters, many of the factors involved are conserved between yeast and human cells.
When first author and HHMI Gilliam Fellow Sheena Vasquez, PhD ’23, began her thesis work as a graduate student, the Drennan Lab and collaborators at BU were attempting to get snapshots of Met18 as part of its full targeting complex in yeast. In the proposed model for the CIA pathway, Met18 forms a complex with two other proteins that deliver iron-sulfur clusters to client proteins.
Capturing Met18 in that complex, however, was proving difficult. In proteins, their shape and orientation can often give clues to the mechanism of their function. The researchers decided to take a step back: what did Met18 look like on its own?
“Our goal was to better understand the proteins that are responsible for delivering iron-sulfur clusters to target proteins in the cell and what the structure of Met18, in particular, could tell us about how it functions,” Vasquez says. “To our surprise, we got some really cool complex structures using a technique called Cryo-Electron Microscopy.”
CryoEM involves isolating proteins in a liquid solution, freezing that sample, and imaging it. The two-dimensional images of proteins from a CryoEM dataset can be used to generate three-dimensional structures.
In isolation, Met18 often interacted with other Met18 proteins, with multiple units twisting together to form a structure called a hexamer, so named because it is made up of six units of Met18.
Drawing on available literature and characterizing protein-protein interactions using pulldown assays, the researchers deduced that certain segments of Met18 have specific roles. One end is a target for degradation, for example. The target for degradation means the cell recognizes this segment rather like a harried parent in a messy playroom—put it away, or I’m throwing it out. A hexamer of Met18 appears to do just that, tucking away the degradation target so it is buried and inaccessible.
Another portion of Met18 can interact with a client protein called Leu1, and another section of Met18 forms bonds with other proteins in the iron-sulfur cluster transfer machinery.
The segments of Met18 that interact with Leu1 and other proteins in the transfer machinery are also tucked away when six units of Met18 are twisted into a hexamer. The researchers chose Leu1 because it’s a metabolically important protein, and it’s easier to work with in solution than some other client proteins that the CIA targeting complex is known to interact with.
Vasquez says it’s possible the hexamer may protect Met18 from being degraded and could also prevent Met18 from transferring clusters prematurely.
Using a different CryoEM sample preparation technique called chameleon, which was new at the time, Vasquez saw something odd: what she initially thought were junk particles were actually Met18 in a different, unexpected state. Instead of a hexamer, it had formed a tetramer—four Met18 units twisted together.
Vasquez says it’s possible that the chameleon method captured a fleeting state of transition, which may give clues to how Met18 prepares itself for delivering sulfur clusters to a client protein. As a tetramer, some of the amino acids of Met18 that bind the client protein Leu1 are a bit more exposed than when Met18 units form a hexamer.
Using mass photometry, the researchers verified that Met18 can form single, double, tetrameric, and hexametric structures. When mixed with a protein known to be part of the CIA targeting complex, Met18 was less likely to form hexamers or tetramers. This could indicate that Met18 responds to the presence of other proteins by disengaging from large, multi-unit structures.
“When it’s not functioning, there could be different states to protect and store itself, and maybe we’re seeing these different states of Met18 preparing itself for its eventual functional unit,” Vasquez explains. “There are a lot of possible explanations. Now that I’m done with this work, I hope these results will be valuable for the field moving forward with other studies.”
CryoEM and Collaborations
Vasquez recently began a postdoctoral fellowship in the Barnes Lab at Sarafan ChEM-H at Stanford University, where she’s drawing on her training in structural biology to study human coronavirus Spike proteins and microbial enzymes that can be utilized for wastewater nitrogen refinement. Throughout her thesis work in the Drennan lab, Vasquez got a front-row seat to the expanding role of CryoEM in structural biology, both in the field and at MIT. The Cryo-Electron Microscopy Facility at MIT.nano was established in 2018, the same year Vasquez started her thesis work in the Drennan Lab.
“It’s very exciting because it’s now a leading technique in the field for obtaining these really interesting structures,” she says. “It definitely taught me a lot, not only about the technique itself but how to work as a team, how to build up a facility, and I’m refining my own knowledge through my current work.”
Co-senior author Deborah Perlstein, PhD ‘05, says wrapping up this project felt like a full circle moment: Perlstein began as a graduate student in Biological Chemistry at MIT at the time Catherine Drennan, the co-senior author on the project, joined MIT as a professor. When Perlstein wanted a deep, molecular understanding of the CIA targeting complex, she naturally thought of a collaboration with Drennan, a leading expert on metalloproteins.
The research was challenging because Met18 is so dynamic–it was difficult to capture a snapshot of its structure because it can alter its conformation to identify and accommodate an expansive number of client proteins. The project began with Amanda Vo, then a graduate student in the Perlstein lab, and Edward Brignole, then a postdoctoral fellow in the Drennan lab; they passed the torch to Vasquez and Marquez, who corresponded often and frequently traveled between the labs at BU and MIT to troubleshoot and bring this project to fruition.
“It is always the unexpected findings that most significantly advance our understanding,” Perlstein says. “It is so gratifying to see so many people’s hard work and unwavering dedication finally pay off.”
Drennan notes that more proteins likely depend on iron-sulfur clusters than are currently known because iron-sulfur clusters will fall apart depending on how protein samples are purified, making them especially difficult to study. The structure of the CIA targeting complex in the process of transferring an iron-sulfur cluster to a client also remains unsolved.
“We have structures of a lot of individual pieces, some of them together, but we don’t know how the cluster interacts with the targeting complex or how clusters are transferred,” Drennan says. “We’ve made enormous progress in the last few years—but there’s still so much more to do.”