Assignment of side-chain conformation using adiabatic energy mapping, freeenergy perturbation, and molecular dynamic simulations

NMR spectroscopic analysis of the C-terminal Kunitz domain fragment (alpha 3(VI)) from the human alpha 3-chain of type VI collagen has revealed that t he side chain of Trp21 exists in two unequally populated conformations. The major conformation (M) is identical to the conformation observed in the X- ray crystallographic structure, while the minor conformation (m) cannot str ucturally be resolved in detail by NMR due to insufficient NOE data. In the present study, we have applied: (1) rigid and adiabatic mapping, (2) free energy simulations, and (3) molecular dynamic simulations to elucidate the structure of the m conformer and to provide a possible pathway of the Trp21 side chain between the two conformers. Adiabatic energy mapping of conform ations of the Trp21 side chain obtained by energy minimization identified t wo energy minima: One corresponding to the conformation of Trp21 observed i n the X-ray crystallographic structure and solution structure of alpha 3(VI ) (the M conformation) and the second corresponding to the m conformation p redicted by NMR spectroscopy. A transition pathway between the M and m conf ormation is suggested. The free-energy difference between the two conformer s obtained by the thermodynamic integration method is calculated to 1.77 +/ - 0.7 kcal/mol in favor of the M form, which is in good agreement with NMR results. Structural and dynamic properties of the major and minor conformer s of the alpha 3(VI) molecule were investigated by molecular dynamic. Essen tial dynamics analysis of the two resulting 800 ps trajectories reveals tha t when going from the M to the m conformation only small, localized changes in the protein structure are induced. However, notable differences are obs erved in the mobility of the binding loop (residues Thr13-Ile18), which is more flexible in the m conformation than in the M conformation. This sugges ts that the reorientation of Trp21 might influence the inhibitory activity against trypsin, despite the relative large distance between the binding lo op and Trp21.