Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering.

The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C(γ) ring pucker preference and the cis-trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple-stranded antiparallel β-sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C(γ) ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C(γ) -endo ring pucker in the native structure. Pro37 was replaced with C(γ) -exo biased pucker derivatives: (2S,4R)-4-hydroxyproline (4R-Hyp), (2S,4R)-4-fluoroproline (4R-Flp), (2S,4R)-4-methoxyproline (4R-Mop), and C(γ) -endo biased pucker derivatives: (2S,4S)-4-hydroxyproline (4S-hyp), (2S,4S)-4-fluoroproline (4S-flp), (2S,4S)-4-methoxyproline (4S-mop) to examine how a preorganized pucker affects the folding stability and ligand-binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S-flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C - F group. Analysis of ligand-binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S-flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4-substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical.