NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

Abstract

The emergence of drug-resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X-ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.

Figure 1

NMR solution structure of PvFKBD. (A) Backbone ensemble of 20 conformers with lowest energy is shown superposed. (B) Ribbon drawing of FKBD of PvFKBP35 (PDB ID: 2KI3). (C) An overlay of the backbone heavy atom trace (N, C′, Cα) of the NMR structures of HsFKBP12 (Blue), PfFKBD (green), PvFKBD (Red) is shown. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

X-ray crystallographic structure of FKBD domain of P. vivax in complex with FK506. (A) The overall structure in complex with FK506 is shown as a ribbon diagram. FK506 ligand is shown with residue side chains depicted in a stick representation (carbon colored grey, nitrogen colored blue, and oxygen colored red). (B) The 2F_o-F_c electron density map encountered at the 1.0 σ level at the region of ligand FK506. The residues near the FK506 are labeled and displayed as stick model in element colors (green, C; blue, N; red, O). Hydrogen bonds are represented by black dashed lines. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

FK506 binding site of PvFKBD. (A) Electrostatic surface representation of the residues near the FK506 binding sites in PvFKBD. The position of bound FK506 is shown as stick model in element colors (yellow, C; blue, N; red, O). Positive and negative charges are in blue and red, respectively. (B) Superimposition of the FK506 binding site of PvFKBD and PfFKBD. Residues of PvFKBD and PfFKBD are labeled and colored in green and red respectively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Superimposition of the backbone traces of apoenzyme PvFKBD colored in pink (β3-β4 and β5-β6 loops colored in gray) and FK506-bound PvFKBD colored in green (β3-β4 and β5-β6 loops colored in red). Regions showing differences are highlighted with arrows and labeled. FK506 ligand is shown as yellow sticks. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5

NMR chemical shift perturbation analysis of PvFKBD upon complex formation with FK506. (A) Overlay of 2D ¹H-¹⁵N spectra of ¹⁵N- labeled PvFKBD in the absence (blue) and the presence (red) of non-labeled FK506 at a molar ratio (PvFKBD:FK506) of 1:2. (B) Averages of the ¹H and ¹⁵N chemical shift changes on the two spectra in panel A, (δ) plotted against the residue number. (C) Mapping of the residues (yellow) located in and near binding pocket and involve in interaction with FK506 (shown as stick in blue colour) on the 3Dstructure of PvFKBD (Protein Data Bank entry 2KI3). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

(A) Heteronuclear ¹H-¹⁵N NOE for the backbone amides of PvFKBD. Backbone ¹H-¹⁵N NOE values as functions of residue number for PvFKBD were measured. The NOE values for free PvFKBD and complexed with FK506 are colored blue and red, respectively. The regions showing backbone dynamics changes are labeled. (B) Sections of the regions β3/β4, α1/β5, and β5/β6 which show backbone dynamics changes upon FK506 binding. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7

Inhibitory effect of PvFKBD on the phosphatase activity of calcineurin. Calcineurin-inhibitory activity by PvFKBD was examined in the absence or presence of FK506. For comparison, PfFKBD and HsFKBP12 were also used in the assay. The calcineurin activity was measured by observing the release of phosphate. In the assay, 5 μM of proteins and 10 μM of FK506 were used. The phosphatase activities represent averages of three independent experiments.

Figure 8

Structural differences in binding pockets of human and parasite FK506 binding domains. (A) The hydrophobic residues (H87, I90, and I91) on the loop between β5 and β6 which can interact with methyl group present on the C11 position of FK506 are shown. Human FKBP12 crystal structure with FK506 (PDBID: 1FKJ) was utilized to highlight the interaction pattern. (B) The variant amino acid residues in PvFKBD and PfFKBD (C105/106, S108/109, and I109/110, respectively) which replaces the H87, I90, and I91 of human FKBP12 are highlighted with sticks. Human FKBP12, PvFKBD, PfFKBD are displayed in green, blue, and magenta, respectively. FK506 ligand and the residues are indicated with sticks. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]