Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

► Prolyl oligopeptidase (PREP) is an important drug target; the active site is on the surface of internal cavity of protein. ► Molecular dynamics (MD) simulation indicates that the inter-domain loop structure is the pathway to the active site. ► Enzyme kinetics study of PREP variants also supports the hypothesis obtained from MD results. ► Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation. ► MD results show that H-bond network holding the inter-domain loop structure in place altered when active site bound.