Cryo-EM structures of Mycobacterium tuberculosis imidazole glycerol phosphate dehydratase in the apo state and in the presence of small molecules

Abstract

Unlike humans, Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, has a de novo histidine-biosynthesis pathway. The enzyme imidazole glycerol phosphate dehydratase (IGPD), which catalyses the conversion of imidazole glycerol phosphate to imidazole acetol phosphate, has been studied extensively from various organisms and has become a major target for the development of antibacterial, antiweed and antifungal small molecules. In our previous studies, we have shown that in crystals IGPD forms a 24-mer oligomeric state in which the monomers are arranged in 432 symmetry. In order to gain insights into the oligomeric state of Mtb IGPD in solution, we determined cryogenic sample electron microscopy (cryo-EM) structures of apo IGPD at 2.2 and 3.1 Å resolution. In addition, we also determined the cryo-EM structure of IGPD in the presence of 3-amino-1,2,4-triazole (ATZ) to 2.8 Å resolution. The results of this work, which corroborate those from the crystallographic studies, indicate that IGPD forms a homo-oligomeric structure in solution comprising of 24 subunits. ATZ binds in the active-site pocket of the enzyme, which is located at the interface of three monomers and tethers 24 ATZ molecules. The results of this study suggest that cryo-EM, in addition to being a rapidly evolving and complementary imaging technology for elucidating 3D structures of biological macromolecules, can be useful in pinpointing the mode of binding small molecules of low mass (here ∼85 Da) and mapping protein-ligand interactions, which could assist in the design of accurate (high-potency) inhibitors.

Keywords: cryo-EM; drug discovery; histidine-biosynthesis pathway; imidazole glycerol phosphate dehydratase; tuberculosis.

Figure 1

Cryo-EM structure of the Mtb IGPD–ATZ structure. (a) The final sharpened cryo-EM map of Mtb IGPD with bound ATZ. The map is coloured in a rainbow using the model. (b) A difference map showing the density of ATZ (red) in the apo map (grey). The difference map was obtained by subtracting the apo map from the ATZ-bound map in Chimera (no normalization was performed). (c) The cartoon representation of the 24-meric assembly of IGPD coloured as in (a). Each monomer within the assembly is visualized to highlight its secondary-structural elements, where α-helices are shown as smooth ribbons and β-strands are represented as flat arrows indicating the direction of the polypeptide chain. (d) Monomer A (brown), monomer B (marine) and monomer C (green) make up the active-site unit with two Mn atoms (Mn1 and Mn2, purple spheres). ATZ is shown in stick representation,

Figure 2

Active-site density of Mtb IGPD. In (a), (b) and (c) the density around the metal-binding sites in apo IGPD, ATZ–IGPD and apo GPD (higher resolution) is shown with the enzyme in stick representation (with C atoms in green) and manganese (purple) and water (red) in sphere representation. The residues coordinating the metals are labelled and are from two neighbouring chains. The C atoms of the ligand ATZ in (b) is shown in yellow. The maps were contoured at 3.5σ in (a) and (b) and at 3σ in (c) in PyMOL. To highlight the residual density in the data set of apo IGPD (higher resolution), in (d) an F_o − F_c normalized difference map (magenta) from Servalcat is shown at 4σ at the active site. This extra unmodelled density is marked with a black arrow in (c).

Figure 3

Structural superimposition of active-site structures derived from cryo-EM and X-ray structures. (a) Structural superimposition of the active site of the apo IGPD cryo-EM structure (PDB entry 9m2p) and the crystal structure (PDB entry 6khh). The cryo-EM structure is represented in green, while the crystal structure is shown in teal, illustrating the alignment and structural similarities between the two forms. Manganese ions Mn1 and Mn2 along with the respective water molecules W1 and W2 of the cryo-EM and X-ray structures are illustrated as spheres in the same colours as described above. (b) Structural superimposition of the active site of the IGPD–ATZ cryo-EM structure (PDB entry 9m2q) and the crystal structure (PDB entry 4lpf). The cryo-EM model is represented in red, while the crystal structure is depicted in yellow. ATZ in the cryo-EM and crystal structures is shown in stick representation in green and blue, respectively. (c) Structural superimposition of the active site of the lower and higher resolution apo IGPD cryo-EM structures (PDB entries 9m2p and 9m2r, respectively), along with the crystal structure of apo IGPD (PDB entry 6khh). The cryo-EM structures PDB entries 9m2p and 9m2r are depicted in green and marine, respectively, and the crystal structure is represented in teal. Additionally, water molecules (W1 and W2) were modelled in the higher resolution apo IGPD structure, offering insights into their interactions with Mn atoms within the catalytic core.

Figure 4

Superimposition of the active-site regions of cryo-EM and crystal structures of IGPD–ATZ complexes. The ATZ molecules bound in the crystal (PDB entry 4lpf) and cryo-EM (PDB entry 9m2q) structures are shown as stick models with C atoms in yellow and red, respectively. In both cases, the N, O and S atoms are shown in blue, orange and deep yellow, respectively. The Mn1 and Mn2 atoms, shown as spheres, interact with the N1 and N4 atoms of the 1,2,4-triazole ring of the ATZ molecules (interactions shown as black dashed lines) in a similar orientation to the crystal structure.