Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: A target for antifungals

Abstract

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

Keywords: Cryptococcus; basidiomycetes; cryoelectron microscopy; fungal pathogens; trehalose.

Fig. 1.

T6P synthase (Tps1) from C. neoformans self-associates. (A) Schematic of the canonical trehalose biosynthesis pathway in fungi. (B) Bacterial two-hybrid results indicate a self-association of LexA-CnTps1_56-669 by a reduction in β-galactoside activity after induction of expression with 1 mM IPTG. Error bars represent SE of triplicate biological replicates (N = 3). Statistically significant differences were demonstrated with an unpaired Student’s t test (****P < 0.0001). (C) Anti-LexA western blot confirms the expression of LexA-CnTps1_56-669.

Fig. 2.

Structure of the unliganded C. neoformans Tps1 homotetramer. (A) Structure of the unliganded C. neoformans Tps1 homotetramer. The density map is shown in gray, overlayed with the model as a ribbon diagram. The protomers are colored and labeled in light blue, green, navy, and magenta. The dimensions of the “front” view of the tetramer are labeled in Å. (B) Structure of the tetramer viewed after a 90° rotation around the vertical axis. (C) Structure of the tetramer viewed after a 90° rotation around the horizontal axis. (D) Ribbon diagram of a single protomer of the unliganded CnTps1 cryo-EM structure with the N and C termini labeled. (E) Density for unliganded CnTps1 is shown in gray.

Fig. 3.

Structure of the C. neoformans Tps1 homotetramer bound to UDP and G6P. (A) Structure of the C. neoformans Tps1 homotetramer bound to UDP and G6P. The density map is shown in gray, overlayed with the model as a ribbon diagram. The protomers are colored and labeled in light blue, green, navy, and magenta. The dimensions of the front view of the tetramer are labeled in Å. Ligands UDP and G6P are shown with a space-filling representation. (B) Structure of the tetramer bound to UDP and G6P viewed after a 90° rotation around the vertical axis. (C) Structure of the tetramer bound to UDP and G6P viewed after a 90° rotation around the horizontal axis. (D) Ribbon diagram of a single protomer of the CnTps1–UDP–G6P structure with the N and C termini labeled and ligands in the substrate-binding pocket shown as atom-colored, space-filling molecules. (E) Density for CnTps1–UDP–G6P is shown in gray.

Fig. 4.

The binding of UDP and G6P induces a conformational change in CnTps1. (A) Overlay of the unliganded CnTps1 protomer (light blue) with the CnTps1 protomer bound to UDP and G6P (green). Density (gray mesh) is shown around ligands UDP and G6P in the catalytic pocket. Position of α-helix 2 is indicated with a black box. (B) Overlay of the unliganded CnTps1 protomer (light blue) with CnTps1–UDP–G6P (green) viewed after a 90° rotation around the horizontal axis. Electron microscopy density is shown for UDP and G6P in the substrate-binding pocket. The position of α-helix 2 is boxed, with the black line demonstrating the movement of approximately 7 Å.

Fig. 5.

Substrate-binding residues of CnTps1. (A) Cryo-EM density for UDP and G6P, shown as light gray mesh. UDP and G6P are shown as atom-colored sticks. (B) View of the CnTps1 residues involved in binding UDP. UDP and residues of the C-terminal domain are shown as atom-colored sticks. Hydrogen bonds are shown by dashes. Key atoms of the UDP molecule are labeled. (C) View of CnTps1 residue R453, which binds G6P. G6P and CnTps1 R453 are shown as atom-colored sticks. Hydrogen bonds are shown by dashes. (D) Relative activity of wild-type CnTps1 and UDP-binding mutants. Error bars represent the SE of three independent measurements. (E) Relative activity of wild-type CnTps1 and the G6P-binding mutant R453A. Error bars represent the SE of three independent measurements.

Fig. 6.

The CnTps1 substrate-binding pocket is highly specific for UDPG. (A) Relative activity of wild-type CnTps1 utilizing either 1 mM UDPG or 1 mM UDP-Gal or UDP as substrates. Error bars represent the SE of three independent measurements. (B) Relative activity of wild-type CnTps1 with 1 mM UDPG and increasing concentrations of UDP-Gal. Error bars represent the SE of three independent measurements. 1 mM G6P is present in all experiments. (C) Shown are the extracted ion chromatograms of [M-H]⁻ at m/z 421.08 for the formation of a disaccharide-phosphate (D-P). (D) Shown are the extracted ion chromatograms of [M-H]⁻ at m/z 565.05 for UDPG and UDP-Gal at 0 and 4 min postcatalysis.