Reconciling the Solution and X-ray Structures of the Villin Headpiece Helical Subdomain: Molecular Dynamics Simulations and Double Mutant Cycles Reveal a Stabilizing Cation-Pi Interaction

摘要

The 36 residue helical subdomain of the villin headpiece, HP36, is one of the smallest cooperatively folded proteins, folding on the microsecond timescale. The domain is an extraordinarily popular model system for both experimental and computational studies of protein folding. The structure of HP36 has been solved using X-ray crystallography and NMR spectroscopy, with the resulting structures exhibiting differences in helix packing, van der Waals contacts and hydrogen bonding. It is important to determine the solution structure of HP36 with as much accuracy as possible since this structure is widely used as a reference for simulations and experiments. We complement the existing data by using all-atom molecular dynamics simulations with explicit solvent to evaluate which of the experimental models is the better representation of HP36 in solution. After 50 ns of simulation initiated with the NMR structure, we observed that the protein spontaneously adopts structures with a backbone conformation, core packing and C-capping motif on the third helix that are more consistent with the crystal structure. We also examined hydrogen bonding and sidechain packing interactions between D44 and R55 and between F47 and R55 respectively, which were observed in the crystal structure but not present in the NMR-based solution structure. Simulations showed large fluctuations in the distance between D44 and R55, while the distance between F47 and R55 remained stable, suggesting the formation of a cation-pi interaction between those residues. Experimental double mutant cycles confirmed that the F47/R55 pair has a larger energetic coupling than the D44/R55 interaction. Overall, these combined experimental and computational studies show that the X-ray crystal structure is the better reference structure for HP36 in solution at neutral pH. Our analysis also shows how detailed molecular dynamics simulations can help bridge the gap between NMR and crystallographic methods.