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BIRS Workshop Lecture Videos

Novel knotted structure Haglund, Ellinor


We discovered hidden complexity in the cysteine-knotted topology of the cytokine leptin characterized by a covalent loop (a so-called zero knot) where part of a terminus is slipknotted through the zero knot. We call this motif a Cysteine Knotted Helical Bundle (CKHB). Up to date, there have been no reports of four-helix bundles with similar threaded topology. We explored the question: Do other proteins contain similar CKHBs? We discovered 11 proteins with similar threaded topology. However, leptin is the only motif with a C-terminal zero knot whereas all other structures have an N-terminal zero knot.Structure-based models were used to in investigate the folding/threading mechanism for six four-helix bundles: four with a threaded topology and two unknotted cytokine homologs. We found that the order of events in folding of the four-helix-bundle is conserved and that the nucleation site for folding is the C-terminal helix. Leptin uses a variation of the same mechanism, but in a unique reversed order in which large structural components of the protein start out as part of the zero knot. This contrasts with the other four-helix bundles, which use large structural components as a scaffold for loop formation. Remarkably, leptin slipknots large structure parts through the C-terminal zero knot while the other CKHBs threads its C-terminal through the N- terminal zero knot like a thread through a needle (a so-called plugging mechanism). Conclusively, since four-helix bundles have similar functional and folding landscape it is important to point out that CHKBs with an N-terminal loop pin down the N-terminal helix (helix A), while leptin has the opposite zero knot, thus keeping the N-terminal dynamic. Crystal structures and modelling of receptor complexes reveals one conserved interface (helix A interacting with the receptor) within the cytokines. This suggests a more dynamic assembly process between leptin and its receptor where the malleability of helix A could affect binding affinity and signaling.

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