Heteronuclear relaxation study of the PH domain of beta-spectrin: restriction of loop motions upon binding inositol trisphosphate

The structural dynamics of protein ligand binding sites is one factor determining the specificity towards related ligands. In this context, the spectrin PH domain, which binds to a number of phosphatidylinositol lipid head groups, was investigated with respect to the dynamics of the binding loops. The latter were found to be of intermediate flexibility on a picosecond to nanosecond time-scale in the free protein and become more rigid upon ligand binding. Significant 15N and proton chemical shift changes occur in the binding loops. The internal correlation time, determined from 15N heteronuclear relaxation data using the standard model-free approach, decreases upon ligand binding. For several residues a concomitant rise in the generalized order parameter is observed. This is interpreted as a dampening effect of the ligand on a slow loop motion, while a fast component is not affected. Molecular dynamics simulations were performed to further investigate this situation. In fact, two time-scales of loop motions in the free state are observed in a 9 ns molecular dynamics trajectory. Agreement with generalized order parameters obtained from the experiment improves when a subtrajectory is analyzed that excludes rare dihedral transitions.