(MEMPHIS, Tenn. – July 13, 2026) Mutations to the protein SPOP are widespread in cancer, yet many remain poorly understood. To address this gap, St. Jude Children's Research Hospital scientists obtained structures of SPOP in both the presence and absence of these mutations. Their work captured the fine balance between active and inactive states of SPOP, showing how its activity is regulated and revealing that a key subset of cancer mutations disrupt this balance, providing the missing clue for their roles in disease. The study was published today in Molecular Cell.
SPOP is a substrate receptor and one part of the larger E3 ubiquitin ligase complex that balances the levels of specific proteins in cells. Those proteins include the key gene-regulators BRD2, BRD3 and BRD4, which contribute to cancer development when their activity or levels are dysregulated. Many SPOP cancer mutations affect how these proteins bind to SPOP, and so their role in cancer can be easily explained. However, mutations in other parts of SPOP are found in some cancers, and their contributions to disease remain an open question.
The researchers, led by corresponding author Tanja Mittag, PhD , Department of Structural Biology , found that individual SPOP molecules assemble into a large ring-like "double-donut" state when inactive. Conversely, a linear "filament" state becomes favored when the protein is activated by its binding partner, Cullin-3. Gain-of-function mutations, which cause SPOP overactivity, exclusively form filaments. Loss-of-function mutations, which hinder SPOP, favor the double donut. This implies that these cancer mutations abnormally tip SPOP's equilibrium either toward the inactive double-donut state or toward the active filament state.
The discovery of the double donut state helps elucidate the role of previously unexplained mutations. The new findings bring prior work from Mittag full circle in understanding how SPOP assembly contributes to cancer.
"SPOP is unique among ubiquitin ligases in that it assembles into long filaments — no other substrate receptor we know of does this — but we didn't understand what this brings to the table," Mittag said. "Now we know that SPOP must assemble into these long filaments to be able to circularize and form the double donuts."
SPOP mutants unbalance inactive donut and active filament equilibrium
The double-donut assembly is made up of two stacked rings, formed by between 22 and 30 individual SPOP molecules, and is wide enough to encircle an entire ribosome. Previous studies have shown how SPOP forms long, thread-like filaments to facilitate binding to substrates. These new structures provide insights into how this activity is regulated, including by showing that Cullin-3, a scaffolding protein within the E3 ubiquitin ligase complex, activates SPOP by driving filament assembly.
"One of the most important roles of the double donut is that it represents an 'off' state; they are essentially an autoinhibited, inactive form of SPOP. The linear filament form is the active state," said co-first author Matt Cuneo, PhD , Department of Structural Biology. "The cancer mutations can bypass this regulation, meaning they are no longer responding to normal cellular signals that switch SPOP between the inactive double-donut structure and the active filament."
In cells, SPOP localizes to membraneless compartments called nuclear speckles. The researchers found that the balance between the double donut and filament was central to this process. "The inactive, double-donut state was strongly associated with nuclear speckles, whereas gain-of-function mutations drove SPOP to the surrounding environment," Mittag said. "This suggests the speckle-associated SPOP is in an off-state, and activation reduces affinity for speckles.
These findings also have potential therapeutic implications. "The newly identified inactive double-donut structure provides a new framework for thinking about how SPOP could be targeted in cancer," said co-first author Mohamed-Raafet Ammar, PhD, Department of Structural Biology. "If we understand the cellular signaling pathways that govern the transition between the inactive double-donut form and the active filament form, we may be able to manipulate SPOP activity in cells for therapeutic benefit."
The discovery of SPOP's inactive double-donut state, its equilibrium with the previously characterized active filament state and the link to nuclear speckle localization set the stage for further investigation into the cellular context of this balance. "The finding was definitely unexpected and took several years and hundreds of hours' worth of access to the cryo-electron microscopy resources at St. Jude to understand," Cuneo said. "But despite all we have learned from these structures, there is still missing information, including highly prevalent cancer mutations, which remain unexplained. This indicates there's still much more to learn."
Authors and funding
The study's other first author is Ömer Güllülü, formerly of St. Jude. The study's other authors are Xinrui Gui, Martin Turk and Brian O'Flynn, of St. Jude; and Kelly Churion and Nafiseh Sabri, formerly of St. Jude.
The study was supported by the National Institutes of Health (R01GM112846 and R01CA301513) and the American Lebanese Syrian Associated Charities (ALSAC), the fundraising and awareness organization of St. Jude.
