The Unique Cysteine Knot Regulates the Pleotropic Hormone Leptin

Abstract

Leptin plays a key role in regulating energy intake/expenditure, metabolism and hypertension. It folds into a four-helix bundle that binds to the extracellular receptor to initiate signaling. Our work on leptin revealed a hidden complexity in the formation of a previously un-described, cysteine-knotted topology in leptin. We hypothesized that this unique topology could offer new mechanisms in regulating the protein activity. A combination of in silico simulation and in vitro experiments was used to probe the role of the knotted topology introduced by the disulphide-bridge on leptin folding and function. Our results surprisingly show that the free energy landscape is conserved between knotted and unknotted protein, however the additional complexity added by the knot formation is structurally important. Native state analyses led to the discovery that the disulphide-bond plays an important role in receptor binding and thus mediate biological activity by local motions on distal receptor-binding sites, far removed from the disulphide-bridge. Thus, the disulphide-bridge appears to function as a point of tension that allows dissipation of stress at a distance in leptin.

Pierced Lasso Topology Controls Function in Leptin

“…As the III receptor-binding site is located in the middle of a very flexible loop area between helices A and B, where there are no coordinates in the crystal structure, thus creating a gap (residues 25−38), the results for this region are based on our reconstruction filling the gap. 18 Our NSD for the bound state shows that the Zero-knot protein has decreased dynamics in the first two eigenvectors at the N-terminus and in the covalent loop, and suggests that receptor-binding site III is affected. Taken together, unthreading the protein backbone probably perturbs the crosstalk between the receptor-binding sites II and III in the bound monomeric state, thus perturbing the dimer interface and lowering the biological activity.…”

“…18 Hence, the observed broader TS in the case of both wt +5 and wild-type proteins can be attributed to these unsuccessful unfolding attempts, expanding R g in this region. 18 In the case of the Zero-knot, the chain is never threaded, so a narrower TS is expected. As the covalent loop is larger in the N-loop protein and the "knot" is shallower, 15 residues cross the backbone compared to 45 residues in the wild-type and wt +5 proteins, and a "trapped" unfolded but threaded state is not observed.…”

“…18 The disulphide bridge can easily be manipulated by the chemical environment and can act as an on/off switch to control the threaded motif. 20 Polymorphism in bovine leptin shows genetic variations at position number 4, where the wildtype arginine is spontaneously substituted with a cysteine.…”

“…18 This broadening of the TS was attributed to a trapped, unfolded but threaded conformation, which does not fully unfold, thus collapsing back to the native state instead of the fully unfolded and unthreaded state. 18 Hence, the observed broader TS in the case of both wt +5 and wild-type proteins can be attributed to these unsuccessful unfolding attempts, expanding R g in this region. 18 In the case of the Zero-knot, the chain is never threaded, so a narrower TS is expected.…”

“…The population of an unfolded but threaded state also affects the shape of the free-energy profile, flattening the TS around a Q CA of 0.5−0.7 for the wild-type and wt +5 proteins ( Figure 4A). 18 Each protein was simulated at its individual equilibrium temperature (at the folding T f , shown in Figure 4A). The plot shows a robust folding free-energy landscape, with only minor changes of the barrier height, with about 2k B T between all proteins (F(Q)/k B T = 6.1 for the wild-type protein, 6.4 for the wt +5 protein, 7.0 for the Zero-knot protein, and the 8.2k B T for the N-loop protein).…”