Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Abstract

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

Fig. 1.

The structure of PfA-M17. (A) Diagram of the unbound PfA-M17 monomer colored by domain: N-terminal domain (Green), helical region linking the two domains (Yellow) and C-terminal catalytic domain (Red). The partially disordered loop that sits at the entrance to the active site chamber in the hexameric assembly is shown in magenta. The single zinc atom present in the site 2 position (the site 1 position is unoccupied in this structure, ) is shown as a black sphere and is labeled. (B, C) Cartoon diagrams of the biologically functional PfA-M17 hexamer colored by chain: A (Green); B (Blue); C (Red); D (Yellow) E (Orange) F (Brown). The six active sites line an interior cavity. (D) The molecular surface of by chain (as colored in (B, C). The active site zinc and carbonate of chain B are visible (Purple spheres). Chains C & D are occluded in this view. The position of the loop (with the molecular surface omitted) in chain B that sits at the entrance to the catalytic cavity is shown by yellow coil (residues 246-265). This region is disordered in the other chains.

Fig. 2.

Stereo diagram of inhibitors binding to active site of PfA-M17. (A) 2.0 Å PfA-M17-BES and (B) 2.6 Å PfA-M17-Co4 active site showing inhibitors bound in the active site. Inhibitors (BES/Co4) are colored in magenta. Carbon atoms of residues are colored green. Zinc is shown as black sphere; Magnesium as yellow sphere and water molecules are light-blue spheres. Hydrogen and metallo-bonds are indicated (dashed lines). Electron density is a composite omit map showing averaged density of 12 protein chains contoured at 3.0 σ calculated by using a model containing only PfA-M17 atoms (no metal, ligand, or water was included in calculation).

Fig. 3.

Comparison of the active site cleft and S1 binding pockets of PfA-M17 and PfA-M1. (A), (B) CCP4MG electrostatic potential surface of the active sites of (A) PfA-M17-BES and (B) PfA-M17-Co4. (C) Cartoon of PfA-M17-Co4 showing the residues lining the S1 pocket (labeled and in stick). (D), (E) show a CCP4MG electrostatic potential surface of the active site of PfA-M1-BES (D) and PfA-M1-Co4 (E). (F) shows a cartoon depicting the residues lining the S1 pocket (labeled and in stick) for PfA-M1-Co4. Throughout this figure, electrostatic surfaces are color-coded according to electrostatic potential calculated by the Poisson-Boltzmann solver within CCPMG. Metal ions are shown as black spheres. The carbonate ion in PfA-M17 (A), (B) is shown as ball and stick (Black). The S1 pocket of active sites are indicated (*).