Structural and Functional Basis of Potent Inhibition of Leishmanial Leucine Aminopeptidase by Peptidomimetics

Abstract

A leucine aminopeptidase primarily hydrolyzes amino acid leucine from the N-terminus end of proteins and is involved in free amino acid regulation, which makes it a potential therapeutic target against neglected tropical diseases including leishmaniasis. We here report the purification and characterization of the leucine aminopeptidase from Leishmania donovani (LdLAP). Using a set of biophysical and biochemical methods, we demonstrate that this enzyme was properly folded after expression in a bacterial system and catalytically active when supplemented with divalent metal cofactors with synthetic fluorogenic peptides. Subsequently, enzymatic inhibition assay denoted that LdLAP activity was inhibited by peptidomimetics, particularly actinonin, which caused potent inhibition and exhibited stronger binding association with the LdLAP. Stronger association of actinonin with the LdLAP was due to a stable complex formation mostly mediated by hydrogen bonding with catalytic and substrate-binding residues in the C-terminal catalytic domain. With molecular dynamics simulation studies, we demonstrate that peptidomimetics retain their topological space in the LdLAP catalytic pocket and form a stable complex. These results expand the current knowledge of aminopeptidase biochemistry and highlight that specific actinonin or peptidomimetic-based inhibitors may emerge as leads to combat leishmaniasis.

Figure 1

Sequence analysis of the LdLAP. (A) Domain architecture of the LdLAP displaying its two domains, i.e., the N-terminal domain (NTD, blue) and the C-terminal catalytic domain (CTCD, orange) with active site residues as orange squares. (B) Alignment of the LdLAP sequence with various LAP sequences depicting a conserved catalytic domain and active site residues (K325, D330, D348, D407, and E409) that are presented as blue stars. Conserved and similar residues are shown in red and yellow boxes, respectively.

Figure 2

Structural characteristics of the LdLAP. (A) Cartoon representation of the modeled structure of the LdLAP. The active site residues are depicted as sticks along with zinc ions as green spheres. (B) Secondary structure of the modeled LdLAP. Cylinders and arrows are representative of helices and sheets, respectively. (C) Ribbon representation of the hexameric structure of the LdLAP. Each monomeric unit is colored differently, whereas two metal ions of each subunit are shown as gray spheres.

Figure 3

Purification and biophysical characterization. (A) SDS-PAGE gel showing the purified LdLAP in lane 1, while lane M presents a prestained protein marker. (B–C) Far-UV CD spectra of the LdLAP with and without metal cofactors and at different pHs. (D) Heat denaturation plots of the LdLAP.

Figure 4

Enzyme activity and inhibition. (A) Michaelis–Menten fit for the amidolytic activity of the LdLAP. Data represents mean activity ± SD (N = 3). (B) Enzyme assay of the LdLAP with different fluorogenic peptides. (C, E, and G) Double-reciprocal plots demonstrating competitive inhibition of the LdLAP with peptidomimetics, while chemical structures of peptidomimetics are presented in the insets of figures. (D, F, and H) Determination of the inhibition constant for actinonin, bestatin, and amastatin.

Figure 5

Peptidomimetics showing high binding association for the LdLAP. (A–F) Fluorescence spectra of the LdLAP with two potent inhibitors actinonin and bestatin along with the Stern–Volmer and modified Stern–Volmer plots showing binding association of peptidomimetics with the leishmanial LAP.

Figure 6

Molecular docking. (A,B) Surface and atomic interactions of peptidomimetic inhibitors with the LdLAP. Inhibitors and interacting residues are labeled and displayed as sticks. Metal ions and polar contacts are shown as gray spheres and black dashed lines, respectively.

Figure 7

MD simulation. Group parameters RMSD (A), RMSF (B), and Rg (C) highlighting that peptidomimetic inhibitors form a stable complex with the LdLAP. Residues are numbered as per the full-length LdLAP sequence in Figure 7B, whereas catalytic residues are denoted by green downward arrows.