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 wildtype 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.

Knotted proteins: Tie etiquette in structural biology

“…It has been proposed that knotted proteins have enhanced thermal (20,26), mechanical (26,27,128) and structural (127) stabilities. Another possibility is that knots help shape and stabilize the active site of enzyme (129)(130)(131), and in some cases it has even been suggested that knots can control enzymatic (130) and signaling activity (132). A recent study put forward the view that knots can impair protein degradation by ATP-dependent proteases leading to partially degraded products with potentially new functions (133).…”

Searching the optimal folding routes of a Complex Lasso protein

“…To complement the picture obtained by means of the EFM we have performed a set of folding simulations employing the well-established GōM proposed by Clementi et al (26), that has already been used by Haglund et al to study lasso proteins (20,22,23). For details on the description we refer to the cited references, here we just underline that the native contacts establish through a 12-10 Lennard Jones potential, which is the main driving force of the folding.…”

“…Since Leptin was classified as the first CL protein(20), this topological state has been found to be widespread in the known PDB structures, characterizing about 18% of the proteins containing a cysteine bridge(21). Most of the CLs are secreted proteins, with signaling functions, and their topology is believed to have a crucial role in their biological activity (22,23). Moreover, the topology of CLs can be controlled externally, since the cysteine bridge is stable in an oxidizing solution, while it does not form in a reducing environment.…”

“…This picture assumes that folding is dominated by native contact interactions while non-native interactions play a minor role(30). GōMs have been validated using both experimental data and more detailed simulation models(31-36), and their predictions are considered reliable in the framework of small protein folding.GōMs have been widely used to study the folding of entangled proteins, providing valuable indications on their thermodynamics and kinetics (37-41), also in the framework of lasso folding (20,22,23). However, the underlying theory clashes with the presence of knots in proteins, as the formation of entanglements implies a high degree of coordination at different length scales which can hardly be encoded in native contact potentials.…”

“…GōMs have been widely used to study the folding of entangled proteins, providing valuable indications on their thermodynamics and kinetics (37-41), also in the framework of lasso folding (20,22,23). However, the underlying theory clashes with the presence of knots in proteins, as the formation of entanglements implies a high degree of coordination at different length scales which can hardly be encoded in native contact potentials.…”