Folding and threading of leptin as a model for pierced lasso topologies.

Abstract

The introduction of entanglements in proteins remains elusive, where the mechanism of formation is unclear. We have for the first time characterized the folding and threading mechanism of a “knot-like” protein. The recently discovered Pierced Lasso Topology (PLT) is a class of proteins in which a disulfide formed covalent loop is threaded by the protein backbone of a free terminus. Within the last decade PLTs have been identified in over 600 known protein structures across all kingdoms of life. These proteins vary in function from oxidoreductases to hydrolases and small hormones. Their presence in different cellular compartments is highly influenced by the redox potential of the environment acting as a potential molecular switch. A combination of experimental phi-value analysis and structure-based models were used to identify key residues within the folding pathway for the model human hormone leptin. Previous computational work uncovered that the threading event occurs within the early stage of the folding free energy landscape. Incorporation of a threading event adds ruggedness to the folding landscape, slowing the process. Our investigation of leptin's in vitro threading event utilized a variant designed to lock the structure in a pre-threaded conformation inhibiting unthreading of the chain in the denatured state. The designed variant folds 100 times faster than the wild type, demonstrating that leptin spontaneously threads/unthreads in oxidizing environments; with the covalent loop intact.