Differential responses of the backbone and side-chain conformational dynamics in FKBP12 upon binding the transition-state analog FK506: implications for transition-state stabilization and target protein recognition.

FKBP12 serves a dual role as a peptidyl-prolyl cis-trans isomerase and as a modulator of several cell signaling pathways. The macrolide FK506 is a transition-state analog of the catalyzed reaction and displaces FKBP12 from its natural target proteins. We compared the conformational exchange dynamics of the backbone and methyl-bearing side chains of FKBP12 in the free and FK506-bound states using NMR relaxation-dispersion experiments. Our results show that the free enzyme exchanges between the ground state and an excited state that resembles the ligand-bound state or Michaelis complex. In FK506-bound FKBP12, the backbone is confined to a single conformation, while conformational exchange prevails for many methyl groups. The residual side-chain dynamics in the transition-state analog-bound state suggests that the transition-state ensemble involves multiple conformations, a finding that challenges the long-standing concept of conformational restriction in the transition-state complex. Furthermore, exchange between alternative conformations is observed in the bound state for an extended network of methyl groups that includes locations remote from the active site. Several of these locations are known to be important for interactions with cellular target proteins, including calcineurin and the ryanodine receptor, suggesting that the conformational heterogeneity might play a role in the promiscuous binding of FKBP12 to different targets.