Structure of a sparsely populated chimeric intermediate that facilitates fold-switching of a metamorphic protein

Metamorphic proteins challenge the single-sequence-structure-function paradigm by switching between distinct folds that often adopt complementary functions. However, the conformational states sampled along the fold-switching pathway have remained elusive because they are transient and unstable. Using multinuclear chemical exchange saturation transfer and relaxation dispersion NMR, combined with chemical shift–based structure determination and protein engineering, we visualized the atomic resolution structure of an “invisible” state of the metamorphic chemokine lymphotactin. This state is a chimera that integrates the secondary and tertiary structure of one fold with the quaternary assembly of the other. Locking lymphotactin into a single conformation abolishes access to this state, supporting its role as an on-pathway intermediate in metamorphosis. The stabilization of such hybrid intermediates is likely to have facilitated the evolution of fold-switching and represents a powerful strategy for protein design.