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Shape Matters: Cells’ Ability to Transport an Essential Protein Depends on its 3-D Conformation

11/06/17

Researchers reveal that the protein RCC1’s overall structure, not its distinct amino acid sequence, determines whether it’s fast-tracked into nuclei.

PHILADELPHIA -- To perform key tasks, cells use molecular carriers to help shuttle a diverse array of specialized proteins – like a car or truck pulling a cargo trailer – into their nuclei. Although some of these nucleus-bound cargoes are essential for life, others cause disease, and researchers have been looking for ways to disrupt the transport chain that can fuel infection and cancer alike. Now, in a study published October 17 in Nature Communications, Thomas Jefferson University researchers have discovered the essential components of the molecular car-to-cargo connection, which is the first step in designing specific methods to block some diseases.

“A clear molecular picture of the connections involved in the nuclear transport is the best thing that can happen to us in terms of drug development,” says senior author Gino Cingolani, PhD, Professor of Biochemistry and Molecular Biology at Thomas Jefferson University, and member of the Sidney Kimmel Cancer Center at Jefferson. “Knowing how these components connect on the molecular level makes it possible to design drugs that interfere with that connection.”

“Most research on this topic has focused on the ‘hitch,’ called the NLS, that connects the car to the cargo,” says Dr. Cingolani. “But our research showed that the molecular shape of the car and the cargo are both critical to creating a strong and specific interaction. It shows us that if we want to break this connection we need to focus on all three components, not just the NLS.”  

The importin a3 molecule, or “car,” is one of a family of importin proteins that help ferry other proteins across the nuclear membrane where they can alter the course of DNA transcription. Most importins, like the importin a1, tow upwards of 500-600 different proteins into the nucleus. In contrast, the importin a3 preferentially ferries just two: RCC1 and NF-kB, two extremely well regulated and important cargoes. In this study, Dr. Cingolani’s “cargo” was the RCC1 protein, which has a multitude of functions in the cell, and without which, the cell dies. 

It makes sense to look beyond the hitch or NLS, explains Dr. Cingolani, since RCC1’s NLS “looks a lot like the NLS attached to other cargos.” But what drives RCC1’s specificity for importin a3, if not a unique NLS sequence? That’s “the thing that puzzled many, many people for a long time,” says Dr. Cingolani.

The answer lies in how the “car,” importin a3, interacts with the three-dimensional structure of the regions that surround RCC1’s hitch, the NLS. To probe the connections between these two proteins, Dr. Cingolani’s team first visualized the crystal structure of RCC1 while bound to importin a3. RCC1 harbors a b-propeller, a bulky molecular region that flanks its NLS. They found that in humans, importin a3 flexibly opens to accommodate the b-propeller, packing it against its lateral surface. Together, RCC1’s shape and importin a3’s ability to change its conformation drive this selective interaction.

In the past, scientists failed to decipher the complexity of the interaction between RCC1 and its importin because they were so focused on RCC1’s NLS. “Nobody, before this structure, had looked at a full-length cargo bound to an importin a,” says Dr. Cingolani.

Dr. Cingolani and colleagues also overlaid the structure of a different member of the importin family, importin a1, onto the structure of importin a3 bound to RCC1, noting that the a1 clashed with RCC1’s b-propeller. Accordingly, Dr. Cingolani’s team observed that RCC1 binds to importin a3 with much higher affinity than a1.

By removing RCC1’s b-propeller or by introducing a short sequence of amino acids between the b-propeller and NLS, researchers eliminated RCC1’s ability to strongly bind importin a3, demonstrating that “the recognition is global; the entire 3-D structure is recognized, not just the NLS,” says Dr. Cingolani.

“Nature gave [RCC1] a dedicated import pathway in humans in order to ferry essential cargos with more complicated shapes and structures,” says Dr. Cingolani. “RCC1 is just such an important protein, that you wouldn’t want it to have to compete with the other thousands of proteins that have an NLS.”

Dr. Cingolani and his team are now investigating specific interactions between importin a3 and its one other main cargo, transcription factor NF-kB, known for its role in cellular inflammation that leads to cancer. “Our preliminary data suggest NF-kB has a complex quaternary structure that, like RCC1, requires an overall structural recognition rather than a simple NLS. We’re pretty positive that we’ll see something similar,” he says.

Now, armed with knowledge of how importin a3 binds its two targets, researchers can design drugs that target this specific import pathway without disrupting other important traffic. “Although many have tried to target and block importin a3, none have been successful so far. Now we have the knowledge to selectively target one specific, unique binding interaction.” 

The study was funded by NIH grants (GM074846, GM100888, and CA56036). The authors report no conflicts of interest.

Article reference: G. Cingolani, et al., “Three-dimensional context rather than NLS amino acid sequence determines importin α subtype specificity for RCC1,” Nature Communications, doi:10.1038/s41467-017-01057-7, 2017.