The Rational Design of Therapeutic Peptides for Aminopeptidase N using a Substrate-Based Approach

Joshi, Shilvi; Chen, Lang; Winter, Michael B.; Lin, Yi-Lun; Yang, Yang; Shapovalova, Mariya; Smith, Paige M.; Liu, Chang; Li, Fang; LeBeau, Aaron M.

Scientific RepoRts | 7: 1424 | DOI:10.1038/s41598-017-01542-5 addition to providing P1 specificity for hAPN, our global analysis revealed numerous non-prime-side specificity features. In particular, these features include, among others, a significant preference for certain hydrophobic residues (such as tryptophan, phenylalanine, and proline) at the P4′ position, a preference for serine/threonine or phenylalanine at the P2′ and P3′ positions, and a decreased preference for proline at the P1′ position (Fig. 1A ,B, and Supplemental Figure 2 ). Structural basis for APN P1 substrate specificity. To provide a molecular basis for APN substrate recognition and catalysis, crystal structures were solved of APN bound to natural free amino acids with varied P1 preferences (methionine, leucine, arginine, glycine, isoleucine, and aspartic acid) to probe the influences of the extended binding pocket. Due to the nearly identical architecture of the active sites and P1 specificities of pAPN and hAPN, pAPN was used for analysis because of its propensity to form high quality crystals 5, 6 . The crystal structure of pAPN in complex with the free amino acid alanine and a seven amino acid poly-alanine peptide substrate were initially determined ( Figure 2 ). The seahorse-shaped ectodomain of pAPN contains 4 domains, head, side, body, and tail ( Fig. 2A) . The N-terminal residue of peptide substrates is firmly anchored in the spacious active site of APN between the head and body domains with residues Gln208, Glu350 and Glu406 forming hydrogen bonds with the free N-terminal amine group (Fig. 2B,C) . The nitrogen of the scissile peptide bond forms a hydrogen bond with the electron-repelling carbonyl oxygen of Ala348, while the carbonyl oxygen of the scissile peptide bond interacts with the electron-attracting zinc and Tyr472 (Fig. 2B,C) . The resonating electrons of the scissile peptide bond are pulled towards the carbonyl oxygen, thus, destabilizing the bond and making it available to nucleophilic attack by the zinc-activated water molecule. At the same time, the activated nitrogen of the scissile peptide bond is also ready to accept a proton from the catalytic water through the side chain of Glu384. In the presence of free alanine, the carbonyl oxygen of Ala348 from pAPN maintains a hydrogen bond with the carboxyl group of the free alanine, suggesting that the carboxyl group oxygen of free alanine near Ala348 is protonated. Protonation of this carboxyl group oxygen of free alanine is due to simultaneous deprotonation of the other carboxyl group oxygen of free alanine by zinc and Tyr472 (Fig. 2D,E) .