Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition

Abstract

The highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall, where multimerization via disulfide formation is unlikely to block the substrate-binding site. Finally, we mapped the regulatory phosphorylation site in thimet oligopeptidase to a location on the outside of the molecule well away from the active site, which indicates this modification has an indirect effect on activity.

Fig. 1.

Molecular surface views of neurolysin (Brown et al. 2001). (A) Two views of neurolysin, rotated by 90° relative to each other, showing the long narrow substrate-binding channel. The active site of the enzyme is located near the floor of the channel. (B) Cutaway view of the channel floor with a model of the bound substrate neurotensin, a 13-residue neuropeptide. This figure and all molecular surface representations were made with the program GRASP (Kraulis 1991).

Fig. 2.

Amino acid sequence alignment of thimet oligopeptidase (TOP) and neurolysin. Sequence differences are highlighted in green. Cysteine residues in TOP thought to be involved in disulfide mediated multimerization of the enzyme are in red, and the site of protein kinase A phosphorylation of TOP is in blue.

Fig. 3.

Surface view of neurolysin showing (blue) positions of surface amino acid differences with thimet oligopeptidase (TOP). (A) View showing the exterior surface of the molecule. (B) Cutaway view of the substrate-binding channel floor with sequence differences labeled. Neurolysin residue numbers are given together with the one-letter codes for neurolysin (before the residue number) and TOP (after the residue number) amino acids. Subtract one from the neurolysin residue numbers for the corresponding residue numbers in TOP. Red labels indicate positions at which the TOP amino acid is conserved in all known TOP orthologs and the neurolysin amino acid is conserved in all neurolysin orthologs. The active site zinc is colored orange. (C) The two walls of the channel with sequence differences labeled.

Fig. 4.

Electrostatic potential at the channel wall surfaces. The wall opposite the active site is on the left and the wall containing the active site on the right. The surface potential was calculated in GRASP (Kraulis 1991).

Fig. 5.

Substrate targeting and sequence differences in thimet oligopeptidase (TOP) and neurolysin. (A) Alignment of three substrates cleaved at different sites by TOP and neurolysin relative to sequence changes in the substrate-binding site. The three substrates are neurotensin (NT) and fluorescently labeled peptides based on portions of the dynorphin A (QF370) and kininogen (Kin) sequences. (B) Amino acids giving the lowest K_m values (Oliveira et al. 2001) at each position in the parent sequence shown aligned relative to sequence differences between TOP and neurolysin in the substrate-binding site.

Fig. 6.

Positions of regulatory sites in thimet oligopeptidase (TOP) mapped to the neurolysin structure. (A) Two 90°-rotated views showing (blue) the positions of the cysteine residues thought to be involved in multimerization of the enzyme. Residue numbers are for neurolysin, with the identity of the neurolysin residue given before the number. Substract one for TOP residue numbers. (B) View of the neurolysin active site showing the nearby cysteine (Cys428) that may be responsible for the sensitivity of both neurolysin and TOP (Cys427) to thiol modifying reagents. The active site zinc (orange sphere) and side-chains of coordinating residues (His474, His478, and Glu503) are shown. This panel was produced with the program RIBBONS (Carson 1987). (C) Position of the TOP serine (residue 644) phosphorylated by protein kinase A.