Mechanism of ligand-induced folding of a natively unfolded helixless variant of rabbit I-BABP.

Substitution of the helix—turn—helix capping motif (residues 9—35) of rabbit I-BABP with a flexible Gly-Gly-Ser-Gly linker results in the loss of stabilizing hydrophobic contacts and renders the β-clamshell structure of this steroidal bile acid transport protein unfolded. However, in the presence of a bile acid ligand, we observe strong coupling between binding and folding, resulting in an enthalpy-driven high-affinity interaction (K A ∼ 4 x 10 5 M -1 ) that "rescues" the native state. We investigate the mechanism of induced folding using fluorescence stopped-flow kinetic measurements to distinguish between conformational selection and induced-fit models. We observe both ligand-dependent and -independent kinetic phases which, together with their relative amplitudes, we attribute to an induced-fit "fly casting" type of model in which transient encounter complexes between the ligand and the extended polypeptide chain may act as nucleation sites for folding. An initial fast ligand-dependent kinetic process appears to be consistent with formation of a hydrophobically collapsed intermediate state which slowly rearranges to a nativelike β-clamshell structure. We show that the intermediate forms at a rate 1000 times slower than the rate of ligand association with wild-type I-BABP, reflecting the large configurational entropic barrier to the coupled binding and folding steps of Δα-I-BABP. We have provided mechanistic insights into how natively disordered states, now commonly identified in biology, may fold on binding a target substrate or ligand.