title: Pierced Lasso Topology Controls Function in Leptin.
abstract: Protein engineering is a powerful tool in drug design and therapeutics, where disulphide bridges are commonly introduced to stabilize proteins. However, these bonds also introduce covalent loops, which are often neglected. These loops may entrap the protein backbone on opposite sides, leading to a "knotted" topology, forming a so-called Pierced Lasso (PL). In this elegant system, the "knot" is held together with a single disulphide bridge where part of the polypeptide chain is threaded through. The size and position of these covalent loops can be manipulated through protein design in vitro, whereas nature uses polymorphism to switch the PL topology. The PL protein leptin shows genetic modification of an N-terminal residue, adding a third cysteine to the same sequence. In an effort to understand the mechanism of threading of these diverse topologies, we designed three loop variants to mimic the polymorphic sequence. This adds elegance to the system under study, as it allows the generation of three possible covalent loops; they are the original wild-type C-terminal loop protein, the fully circularized unthreaded protein, and the N-terminal loop protein, responsible for different lasso topologies. The size of the loop changes the threading mechanism from a slipknotting to a plugging mechanism, with increasing loop size. Interestingly, the ground state of the native protein structure is largely unaffected, but biological assays show that the activity is maximized by properly controlled dynamics in the threaded state. A threaded topology with proper conformational dynamics is important for receptor interaction and activation of the signaling pathways in vivo.
authors.affiliation: Center for Theoretical Biological Physics (CTBP) and Departments of Physics and Astronomy, Chemistry and Biosciences, Rice University , Houston, Texas, United States.
authors.forename: Ellinor
authors.initials: E
authors.lastname: Haglund
authors.affiliation: Department of Chemistry and Biochemistry, The University of California, San Diego (UCSD) , La Jolla, California, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Pilko
authors.affiliation: Department of Chemistry and Biochemistry, The University of California, San Diego (UCSD) , La Jolla, California, United States.
authors.forename: Roy
authors.initials: R
authors.lastname: Wollman
authors.affiliation: Department of Chemistry and Biochemistry, The University of California, San Diego (UCSD) , La Jolla, California, United States.
authors.forename: Patricia Ann
authors.identifier: 0000-0002-7478-2223
authors.initials: PA
authors.lastname: Jennings
authors.affiliation: Center for Theoretical Biological Physics (CTBP) and Departments of Physics and Astronomy, Chemistry and Biosciences, Rice University , Houston, Texas, United States.
authors.forename: José Nelson
authors.initials: JN
authors.lastname: Onuchic
mesh_terms: D020738:Leptin / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D056004:Molecular Dynamics Simulation; D015202:Protein Engineering
chemical_list: D020738:Leptin