Identification and Rationalization of Kinetic Folding Intermediates for a Low-Density Lipoprotein Receptor Ligand-Binding Module.

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time. We demonstrate that misfolded or partially folded disulfide intermediates are indistinguishable from the unfolded state when focusing on the backbone NMR signals, which provide information on the formation of only the final, native state. However, C labeling of cysteine side chains differentiated transient intermediates from the unfolded and native states and reported on disulfide bond formation in real time. The cysteine pairings in a dominant intermediate were identified using C-edited three-dimensional NMR, and coarse-grained molecular dynamics simulations were used to investigate the preference of this disulfide set over other non-native arrangements. The transient population of LA5 species with particular non-native cysteine connectitivies during folding supports the conclusion that cysteine pairing is not random and that there is a bias toward certain structural ensembles during the folding process, even prior to the binding of calcium.