Understanding Knotted Proteins: From Birth to Degradation
WPI-SKCM² Principal Investigator Dr. Shang-Te (Danny) Hsu and his team study topologically knotted proteins, whose polypeptide chains physically thread through themselves, forming knots similar to those you would find on a rope. As a structural biologist, Hsu is looking for these knots and working to uncover how they may affect processes such as protein stability, folding pathways, and cellular function.
Revealing the smallest and most complex protein knot to date
Prior to the advent of AI, researchers relied heavily on technically challenging experimental methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM), to determine the three-dimensional protein structures. Then came Google DeepMind’s AlphaFold, which predicted over 200 million structures from amino acid sequences alone.
Last year, Hsu’s team set out to validate one of AlphaFold’s predictions. Using X-ray crystallography and NMR spectroscopy to determine the protein’s structure, they found a near-perfect match, confirming the presence of a small and tight 7₁-torus knot. This represents the smallest and most complex protein knot identified to date and was published in the Journal of Biological Chemistry.
Read the Google DeepMind highlight, or see the original study in Journal of Biological Chemistry.
Watching a knotted protein fold at birth
At birth, proteins start as long amino acid chains that fold into their active form. Some of this folding begins while the protein is still being synthesized on the ribosome, termed cotranslational folding. Hsu’s group recently focused on a 31 trefoil knotted protein to study its folding pathway and gain insight into the broader problem of cotranslational folding.
The team found that, when bound to a chaperone protein on the ribosome, the nascent chain adopts a long helical structure inside the ribosomal tunnel that does not appear in the protein’s final folded structure.
To capture this intermediate state, the researchers used an engineered arrest peptide that stalls the ribosome, enabling visualization by cryo-EM. In addition to serving as a stalling tool, the engineered peptide itself is studied in the paper, revealing how it functions at the molecular level. These combined findings were recently published in Nucleic Acids Research.
Learn more about the ribosome arrest peptide in Cryo-EM Structure Illuminates How a Designed Peptide Precisely Pauses the Ribosome, and the full study published in Nucleic Acids Research.
Uncovering how a knotted protein works in protein degradation
At the end of their life cycle, proteins marked for degradation are tagged with ubiquitin, a small regulatory protein that directs them to the proteasome, the cell’s recycling machinery. One player in this process is UCHL5, a 52 Gordian knotted protein that removes ubiquitin chains from degradation targets. However, until now, how branched ubiquitin chains are recognized and processed has remained unclear.
In a recent study published in Nature Communications, Hsu and colleagues used cryo-EM to reveal how a K11/K48-branched ubiquitin chain is recognized by the human proteasome. In the process, they found that UCHL5 was difficult to visualize and appeared spatially distant from the proteasome.
They proposed that this is because UCHL5 is loosely tethered via the proteasomal subunit RPN13, allowing UCHL5 to move flexibly and avoid being captured in a fixed position during imaging. Since cryo-EM could not fully resolve the interaction, the team instead applied chemical cross-linking mass spectrometry (XL-MS) to confirm the contacts between ubiquitin and a catalytically dead UCHL5 variant together with its proteasomal binding partner RPN13.
Read more on the Institute of Biological Chemistry website, or see the original study in Nature Communications.
Taken together, these studies illustrate how Hsu is building a comprehensive picture of topologically knotted proteins, a large class that may constitute 1–2% of all natural proteins and whose biological roles we are now starting to unravel.
