Shining a Light Into the Shadow of Protein Structure and Function

A revolution in protein imaging capabilities is allowing biomedical researchers to probe what was previously invisible — hidden-in-plain-sight features, blink-and-you’ll-miss-it moments, and dynamics unfolding on time scales once beyond detection.

By capturing fleeting time frames and forms, St. Jude scientists are gaining new insights into protein function and revealing previously unidentified avenues for therapeutic design. With advances in biomedical engineering and computational analyses to match, St. Jude is well positioned to push the boundaries of discovery in protein sciences.

GPCR intermediate structures reveal the fine tuning of signaling responses

Propelled by a period of microscopy and spectroscopy innovation referred to as the “resolution revolution,” researchers have teased out a richer understanding of some of the most important proteins in the body. This is exemplified by the mechanistic insight gained into G protein-coupled receptors (GPCRs), membrane proteins that are vital to numerous cellular processes, ranging from metabolism to neurotransmitter signaling. Extracellular molecules bind to GPCRs, which activate signaling pathways inside the cell to generate an appropriate response to the bound molecule.

GPCRs are not just on/off switches, however. Modulation of the amplitude of the signal allows for more variable responses. Limited insight into how different molecules produce varying levels of response has hindered rational drug development for GPCRs, even though one-third of Food and Drug Administration (FDA)-approved drugs target these receptors. To find answers, St. Jude researchers examined the protein’s fleeting intermediate steps in detail.

In a study published in Nature, the scientists used time-resolved cryo-electron microscopy, molecular dynamics simulations, and single-molecule fluorescence approaches to catch a glimpse of the states-between-states that GPCRs move through to elicit a response — to observe the intermediate steps of GPCR dynamics after a molecule binds, but before the signal relay concludes.

They successfully detailed how three very different drugs for the m-opioid GPCR fine tune receptor signaling, demonstrating that each molecule moves the GPCR through its intermediate steps in different ways, with strongly binding molecules promoting faster and more efficient transitions than weaker ones.

“We found that for weaker GPCR stimulants, the system slows down as it gets stuck in specific steps while changing shape during activation,” said corresponding author Georgios Skiniotis, PhD, Center of Excellence for Structural Cell Biology director and Department of Structural Biology member. “Regardless of how strongly a binding molecule activates the system, the steps remain the same, but the weaker drugs take longer to move through those steps, correlating with their efficacy.”

The intermediate states described in the study reveal the inner mechanics of GPCR signaling at the atomic level. By knowing these details, scientists can design next-generation drugs that are more refined in modulating signaling and maximize safety while maintaining efficacy.

Hidden kinase mechanism revealed

The ability to capture intermediate protein states in such fine detail allows researchers to observe all the shapes a protein can take, called its “conformational landscape.” Charalampos Babis Kalodimos, PhD, Department of Structural Biology chair, is exposing previously invisible states within the conformational landscape of the second largest group of therapeutic targets: protein kinases.

Kinases are critical regulators of protein function, removing a phosphate group from adenosine triphosphate (ATP) and attaching it to a target protein in a process called phosphorylation. Many biological functions, such as cell migration, rely on processive phosphorylation, in which multiple phosphates are adhered to different sites in a single protein.

Processive phosphorylation requires the kinase to transition between different conformations rapidly. While some states of this conformational landscape are well established, a study from corresponding author Kalodimos, published in Science, used nuclear magnetic resonance spectroscopy to investigate the short-lived intermediate states adopted by members of the Src kinase family.

There are likely other hidden or invisible states that we have not yet detected. We’ve only scratched the surface. Many more invisible states remain to be revealed.

Charalampos Babis Kalodimos, PhD

Department of Structural Biology

The researchers found that Src has a hidden state that acts like a quick-release mechanism, ensuring the by-product of ATP, adenosine diphosphate (ADP), is rapidly discarded from the kinase. This allows the kinase to successively phosphorylate without needing to disengage and reengage the protein each time.

While the fleeting appearance of this hidden state explains its elusiveness, its conservation across other Src-family kinases, such as Lck and Hck, underscores its importance for cell function. When the researchers abolished the state using targeted mutations, they found that cell migration (controlled by Src and Hck) and T-cell regulation (controlled by Lck) were significantly impaired.

The researchers are now exploring hidden states across other kinases to understand this essential protein family better. “There are likely other hidden or invisible states that we have not yet detected,” Kalodimos said. “We’ve only scratched the surface. Many more invisible states remain to be revealed.”

Integrated approach reveals heat-shock protein assembly

Pursuing the elusive within protein conformational landscapes is not restricted to kinases. In a study published in Molecular Cell, corresponding author Kalodimos and his team presented the first full-length structures of two molecular chaperone proteins assembled together, revealing key structural features regulating their function.

Heat-shock proteins are molecular chaperones essential in organisms ranging from bacteria to humans. Two of these proteins, Hsp40 and Hsp70, help incorrectly folded proteins attain their correct shape, thereby promoting cellular homeostasis. Exactly how Hsp40 and Hsp70 work as an assembly to fix misfolded proteins was unknown; no one had been able to overcome the technical hurdles involved in capturing the massive and highly dynamic complex.

Kalodimos and his team combined cryo-electron microscopy, nuclear magnetic resonance spectroscopy, and X-ray crystallography to finally reveal the structure of the assembled bacterial chaperone complex. They captured multiple forms of the assembly, consisting of dimers (pairs) of Hsp70 interacting with dimers of Hsp40, in several different states of association with misfolded proteins.

“We obtained key structures of the active and inhibited states bound to an incorrectly folded protein, but we also solved multiple smaller pieces,” Kalodimos said. “By patching them together, they’ve given us a new model of how the Hsp40 and Hsp70 assembly really functions.”

The researchers paid particular attention to a section of Hsp40 called the G/F region. They found that this region binds the incorrectly folded protein first, then latches onto the Hsp70 active site. Hsp40 then pulls the incorrectly folded protein into the active site, displacing itself, but remaining bound to Hsp70 at a different location. After the handoff, Hsp70 then refolds the incorrectly folded protein.

“We now understand mechanistically how the chaperones and protein clients form this very big assembly, how they are released, and how they cycle through this conformational landscape,” Kalodimos said. “Using that knowledge, we have a starting place to look for therapeutic interventions to compensate for disease-causing mutations, exploit as drug targets in cancers, or inspire next-generation antibiotics.”

Hidden nuclear receptor pocket gives new life to PXR drug discovery

Structural studies have unlocked new therapeutic potential in key targets such as GPCRs, kinases, and heat shock proteins. However, drug design is the first step on a long road to clinical benefit. Once exposed to the internal miasma of a cell, optimized therapeutics may be degraded or removed before they can have the desired effect. This is the role of pregnane X receptor (PXR), a nuclear receptor that binds to potential toxins and drugs, including chemotherapeutics, triggering the production of enzymes responsible for their removal.

Blocking PXR is beneficial for the efficacy of many cancer treatments, but different types of nuclear receptors are structurally similar. This makes it difficult to design a drug to target a particular receptor — precisely the problem Taosheng Chen, PhD, PMP, Department of Chemical Biology & Therapeutics, set out to solve.

“My lab has had great success in developing PXR inhibitors, but a subtle chemical change or a point mutation in PXR can convert an inhibitor to an activator,” explained Chen. “This is dangerous, because the inhibitor itself, if converted to an activator, could not only lose efficacy but have the opposite effect. A different approach, such as a PROTAC that destroys the protein, wouldn’t have this issue.”

PROTACs are bifunctional drugs that bind protein targets with one end and recruit protein-degrading machinery with the other, presenting a promising alternative to inhibitors. However, the deep PXR binding pocket traditionally targeted for small molecule drug discovery has hindered the rational design of effective PROTACs. Chen explored already available PROTACs to determine if any could be repurposed to bind and degrade PXR.

In a study published in Nature Communications, corresponding author Chen and his team identified a potential candidate, MD-224, a PROTAC designed for anticancer therapy. MD-224 effectively degraded PXR but had an unexpected twist: It bound to a previously unnoticed binding pocket, which allowed it to recruit protein-degrading machinery more effectively.

While MD-224 also bound other nuclear receptors, only those that shared close similarities with PXR were affected. This could also be tuned, suggesting it is possible to achieve selectivity. Ultimately, a newly discovered targetable binding pocket offers new avenues for drug discovery.

“Our angle is PXR, but the study is very relevant for those who think about other receptors too,” Chen said. “There are many exciting opportunities for other researchers stemming from what we’ve uncovered for this protein family.”

Structural insight of Fanzor2 nuclease demonstrates new bioengineering potential

Understanding the structural variation among closely related proteins, such as nuclear receptors, allows researchers to understand their unique features better. While Chen explores this protein family for exploitable features in drug discovery, Elizabeth Kellogg, PhD, Department of Structural Biology, has uncovered the potential for a bioengineering breakthrough in an ancient family of DNA-editing proteins called Fanzors.

CRISPR-Cas9 technology is derived from a naturally occurring genome-editing system called an RNA-guided nuclease that bacteria use as a defense mechanism. Its discovery ushered in a new paradigm in genomic engineering capabilities. Recently, another large protein family found in bacteria, called TnpB, was discovered to be a functional, but distinct, predecessor to the Cas12 family of RNA-guided nucleases. The Fanzor1 and Fanzor2 proteins are the eukaryotic counterparts to TnpB and are encoded by transposable elements embedded in the genome.

In a study published in Nature Structural & Molecular Biology, corresponding author Kellogg obtained the structure of Fanzor2 to chart how these systems have evolved, offering key insights into the relationship between structure and function for these RNA-guided nucleases. The work revealed that RNA’s role in structuring the active site of Fanzor2 differs from other similar proteins, suggesting that the RNA and protein co-evolved on a separate evolutionary branch from the Cas12 family.

Kellogg hopes this structure will be a launchpad for engineering the next generation of RNA-guided nucleases. “The structural diversity of these assemblies is something that we have no understanding of at all,” she emphasized. “That’s where I think it’s important, not only to know the functional constraints that make something an RNA-guided nuclease, but also how you apply those principles and harness them in engineering.”

One key factor that makes TnpB and Fanzor proteins so exciting is their size — they are significantly smaller than Cas9 and Cas12. In terms of genome engineering, minimizing the size of the protein offers more functionality. “Fanzor2 is already pretty minimal, but its structure suggests there’s malleability in terms of how it can function with the RNA,” Kellogg said. “It hints that we could reduce its size further, but there’s a lot more to do to understand that.”

It’s important, not only to know the functional constraints that make something an RNA-guided nuclease, but also how you apply those principles and harness them in engineering.

Elizabeth Kellogg, PhD

Department of Structural Biology

AI unlocks potential in tandem CAR T–cell therapy

Capturing the Fanzor2 structure is vital not only for understanding the functional constraints of RNA-guided nucleases, but also for revealing how the design principles that regulate their activity can be applied in bioengineering. While this journey is just beginning, computational advances and artificial intelligence (AI) are taking such bioengineering pursuits to previously unattainable heights.

St. Jude scientists have already shown how AI can revolutionize the design of another bioengineering innovation: chimeric antigen receptor (CAR) T cells. These engineered T cells are a type of immunotherapy that reprograms a patient’s existing immune cells to target a tumor-specific protein marker. While CAR T cells have successfully treated some blood cancers, they have not been as effective in targeting solid and brain tumors. One reason is that cancer cells do not uniformly express the same surface proteins. This means CAR T cells targeting a single antigen can miss malignant cells that do not express that protein.

Scientists have tried to create CARs that target two proteins at once, called tandem CARs. However, they have encountered problems, including poor CAR expression on the surface of T cells and suboptimal cancer-killing ability.

A study published in Molecular Therapy by co-corresponding authors Giedre Krenciute, PhD, Department of Bone Marrow Transplantation & Cellular Therapy, and M. Madan Babu, PhD, FRS, senior vice president of data science, Center of Excellence for Data-Driven Discovery director, and Department of Structural Biology member, set out to address these challenges.

For CAR T cells, surface expression of the CAR is vital to ensure that the cell contacts its target protein on tumor cells. In their pursuit of tandem CARs, the team generated 24 variations, but none of them reached the cell surface. “We repeated everything multiple times to ensure we weren’t missing anything and eventually acknowledged that our approach needed to be adjusted,” Krenciute said. “We needed AI input.”

The team developed and validated an AI-informed computational pipeline that significantly accelerated tandem CAR design with improved surface expression and anti-tumor function. Specifically, the pipeline predicted a better tandem CAR design if targeting two proteins expressed in pediatric brain tumors: B7-H3 and IL-13Rα2. The researchers tested their optimized CAR T cells and compared them to several single-target CARs in mouse-tumor models that reflected the mixture of expression seen in patients: cells with both targets, one target or the other, or neither target.

The AI-informed tandem CAR completely cleared tumors in four out of five mice. In contrast, all tumors treated with CARs targeting a single antigen grew back. The results provide evidence that leveraging AI can help the design of other tandem CARs and be used to develop more efficacious designs more quickly than by manual testing.

“Researchers can use our approach to help screen and create better tandem CARs, bringing us closer to the day we can successfully treat challenging tumors, such as pediatric brain cancers,” Krenciute said.

Continuing to explore biology’s hidden states

As researchers continue to probe the unknown spaces of biological function, novel approaches to treating catastrophic diseases are revealed. The technological leaps taken to uncover unmet potential within proteins, combined with the dawn of AI-informed analysis, have brought once elusive conformational capabilities into full light.

As discoveries change what is known about protein motion and activity, and as novel technologies and innovations in approach make it possible to do this work faster and more efficiently, St. Jude is well poised to continue this important pursuit. Investigators are already working to capture new protein states, pulling back the curtain even more and revealing never-before-seen views into the hidden dynamics of life.