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.

Because pAPN forms the same interactions with a free alanine and the N-terminal alanine residue of a poly-alanine peptide substrate, we believe that the crystal structures of pAPN complexed with free amino acids reflect the interactions between pAPN and the N-terminal amino acid residues of at least simple peptide substrates 4 . All of the free amino acids in the crystal structures bound to pAPN in a similar manner as alanine, with the exception of key differences in their side chain interactions. The amino acid side chains were oriented in a pocket in the pAPN body domain with hydrophobic walls and an open end (Fig. 3A) . The side chains with different lengths were able to fit into the pocket because of the open end and were found to primarily form hydrophobic interactions with the walls of the pocket (Fig. 3B ). For example, C β , C γ , and C ε of the methionine side chain formed hydrophobic interactions with Met349/Gln208, Ala346, and Phe467 on the pocket walls (Fig. 3B ). Polar side chains were also able to form additional interactions with pAPN as in the case of arginine where the guanidine group formed a cationic-Pi interaction with Phe467 (Fig. 3B ). In general, the APN-binding affinity of amino acids is positively associated with the extent of hydrophobic or other affinity-increasing interactions between their side chains and the pocket walls. Consequently, amino acids with long, nonpolar side chains like leucine generally have high APN-binding affinity. Analysis of the crystal structures of pAPN complexed with non-favored amino acids found that those residues all shared one unique feature -their side chains had unfavorable interactions with the catalytically critical carbonyl oxygen of Ala348 from pAPN (Fig. 3B ). For example, the C γ atom of isoleucine was 2.4 Å away from the carbonyl oxygen of Ala348, respectively ( Fig. 3B ). At these short distances, a strong van der waals (VDW) repulsion existed between the atoms. Additionally, the O δ group of aspartic acid formed an unfavorable charge repulsion with the carbonyl oxygen of Ala348 (Fig. 3B) . As a comparison, the side chains and disfavored (Ile and Asp) P1 residues for APN. pAPN residues are in magenta, and amino acids are in green. The catalytically critical hydrogen bond between the carbonyl oxygen of Ala348 and the C-terminal carboxyl group of amino acids is shown as a red dashed line in the structure with Ala, but is omitted in other panels for clarity. The interactions between the carbonyl oxygen of Ala348 and amino acid side chains are shown as black dashed lines. The distance between the carbonyl oxygen of Ala348 and the nearest atom on amino acid side chains is shown as a bidirectional arrow.