Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470

Abstract

Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity invitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

Figure 1. Inhibition of EcMAP2 and HsMAP2 by Fumagillin (A) and TNP470 (B)

MAP2 enzyme ( 0.5μg) was incubated with or without Fumagillin or TNP470 in enzyme dilution buffer (20mM HEPES pH7.5, 40mM NaCl, 50μM CoCl₂, 100 μg/ml BSA, 2%DMSO) for 30min on ice before starting the assay. Assay was performed as described in materials and methods. 50μl of reaction mixture contained 50mM assay buffer, 100μM CoCl₂, 4mM Met-Ala-Ser substrate, 0.2U L-AAO, 0.5U HRP and 1mM o-dianisidine. Reaction was started by adding the enzyme to the assay components, increase in absorbance at 450nm was measured every 5min at 37°C.

Figure 2. Ribbon diagram of E. cuniculi MetAP2

E. cuniculi MetAP2 is shown as a ribbon diagram with the catalytic domain and subdomain insert shown in dark and light pink, respectively. Two metal ions coordinated within the active site of the ‘pita-bread’ fold are represented as silver spheres.

Figure 3. Structural sequence alignment of E. cuniculi MetAP2 with other MetAP family members

Structure-based sequence alignment of E. cuniculi MetAP2 with selected MetAP2 and MetAP1 family members. Numbering on the top is based on the sequence of E. cuniculi MetAP2. Identical and similar residues are on a red background and colored red, respectively. Residues coordinating metal ions and contacting fumagillin/TNP470 are indicated with a blue star and purple triangle, respectively. Protein data bank (PDB) structure coordinate ID's are: Human MAP2 (1BOA), Pyrococcus Furiosus MAP2 (1XGS), Human MAP1 (2GZ5), Escherichia coli MAP1 (1MAT), Mycobacterium tuberculosis MAP1 (1Y1N). Enterocytozoon bieneusi Genebank accession number EED44036 [68].

Figure 4. Superposition of E. cuniculi and Human MetAP2 structures

Ribbon diagram of the superposition of E. cuniculi and human MetAP2 X-ray structures supporting the classification of E. cuniculi MetAP as a type 2 methionine aminopeptidase. E. cuniculi and human MetAP2 are shown in pink and green, respectively and active-site metal ions are depicted as spheres. The human MetAP2 structure coordinates are derived from PDB ID 1BOA.

Figure 5. Metal coordination within the E. cuniculi MetAP2c active site

Residues coordinating two iron ions within the active site are shown as sticks with black dashes representing coordination bonds. Iron ions and water molecules are represented as silver and red spheres, respectively. Bonding distances are in units of angstroms (Å).

Figure 6. E. cuniculi MetAP2c-apo with superimposed L-methionine

The active site of E. cuniculi MetAP2c-apo is shown superimposed with the bound L-methionine from HsMetAP2b (PDB ID 1KQ9). Residues likely involved in catalysis (H109 and H218) and those making up the hydrophobic methionine-binding pocket are shown as pink sticks. L-methionine is shown as a stick with predicted coordination bonds to the iron ions represented as black dashes.

Figure 7. E. cuniculi MetAP2c complexed with fumagillin and TNP-470

(A) Structures of the natural product fumagillin and semi-synthetic product TNP-470. (B) The active site of E. cuniculi MetAP2c with fumagillin covalently bound to H109. (C) TNP-470 covalently bound to H109 in the active site of E. cuniculi MetAP2c. In (B) and (C) residues making covalent, hydrophobic, and hydrogen bonding interactions to fumagillin/TNP-470 are shown as sticks with hydrogen bonds depicted as black dashes and iron ions as spheres. Electron density (2f_o-f_c; 1σ contour) for both inhibitors is shown as red mesh. (D) Superposition of the E. cuniculi MetAP2c-apo (pink) and MetAP2c-fumagillin (blue) structures. The structures have a root-mean-squared deviation of 0.16 Å over 356 α-carbon atoms.

Figure 8. Comparison of the microsporidial MetAP2 and human MetAP2b-fumagillin active sites

(A) Superposition of the fumagillin bound structures of E. cuniculi MetAP2c in blue and HsMetAP2b in magenta. (B) Superposition of the homology model of E. bieneusi MetAP2 in orange and HsMetAP2b-fumagillin structure in magenta.