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.