Fig. 6: The active site and proposed mechanism.
From: Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy
a Close-up of Fig. 5c depicting the final segment (L113–P121) of the PD defined in the final Fourier map as a stick model with yellow carbons and black residue numbers running across the active-site cleft. The likely P1 and P1′–P3′ residues are labelled. In addition, selected residues of the active site are depicted for their side chains with carbons in tan and numbered in light blue. Two solvent residues potentially relevant for catalysis are shown as green spheres. The inset provides a slightly rotated close-up view to highlight the interaction (magenta lines) of K118 with E188, Q173 and a solvent molecule. b Same as (a) depicting the product complex of mature neprosin (crystal form I), with the C-terminal tail from a symmetry mate spanning A403 and the His6-tag residues (H404–H409) as a stick model with carbons in cyan featuring substrate subsites P6–P1. A solvent molecule potentially relevant for catalysis (green sphere) bridges E297 and E188 (magenta sticks). c Same as (b) for crystal form II. The C-terminal tail from a symmetry mate spanning A401 and part of the His6-tag (H404–H408) is shown as a stick model with carbons in plum, probably covering subsites P6–P1′. A solvent molecule potentially relevant for catalysis (green sphere) bridges E297 and E188 (magenta sticks). A second solvent molecule (yellow arrow) probably occupies the position of the scissile carbonyl oxygen in the Michaelis complex. The polypeptide chains of both crystal forms overlap for tag residues H404–H409 (crystal form I) and A401–H406 (crystal form II) upon superposition of the respective CDs. d Model of the likely Michaelis complex between a substrate spanning residues P–Q–P*Q–L–P (green carbons) at positions P3–P3′ and the active site of neprosin. Selected residues are displayed for their side chain (plum carbons) and labelled. The catalytic solvent is depicted as a cyan sphere. e Proposed chemical mechanism of substrate cleavage by neprosin.
