Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome

Abstract

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

Fig. 1. Function and overall structure of human HGSNAT.

a HGSNAT catalyzes the transmembrane acetylation of HS in the lysosome. The disaccharide repeating unit is represented by orange and magenta hexagons, while the terminal GlcN is shown as a green hexagon. Acetyl-CoA is represented by a yellow triangle (acetyl group) connected with a red circle (CoA). b Cryo-EM structure of human HGSNAT bound with acetyl-CoA. The two monomers within the dimer are colored in blue and orange respectively. Acetyl-CoA is colored in green. c Electrostatic potential of HGSNAT dimer. d Structure of HSGNAT dimer. e Topology map of the HGSNAT monomer colored in discrete colors corresponding to (d).

Fig. 2. Substrates binding and reaction mechanism.

a Structure of the substrates complex with the densities of acetyl-CoA in cyan and MU-βGlcN in light green. b Densities and models of acetyl-CoA (cyan) and MU-βGlcN (light green) in monomer B of the substrates complex. c Densities and models of CoA (blue) and MUF-NAG (green) in monomer B of the products complex. d 2D ligand interaction plot for the substrates binding site. e Schematic of the ternary complex and ping-pong mechanisms. A monomer of HGSNAT is represented by the blue rectangle with the active site shown as white circle. f Structure of the substrates binding site. g Structure of the product binding site. Interactions between atoms are shown by dotted lines and distances (in Angstroms) between atoms involved are reported in red. The structure of only one monomer is shown but distances between the equivalent residues for the other monomer are labeled in parenthesis. h Ternary complex mechanism. i Ping-pong mechanism. j Logo plot (in Clustal 2 color scheme) of 4640 species and sequence alignment of 11 representative species in the region where N286, H297, and D307 are located. k Enzymatic activities of HGSNAT mutants. All the enzymatic activities of HGSNAT mutants were normalized to the expression level and shown as % of HGSNAT WT. Results are shown as mean ± SD (n = 3 replicates). Source data are provided as a Source Data file.

Fig. 3. Protein dynamics influence acetyl-CoA accessibility.

a Acetyl-CoA binding pore within TMD shown in surface representation with the electrostatic potential. Only one monomer is shown for simplicity. b, c Sidechain conformation changes gate the acetyl-CoA binding pore. Residues from the Acetyl-CoA structure are colored in green, residues from the Apo_trans structure are colored in brown, and residues from the Apo_ground structures are colored in black. d Cytosolic portion of the acetyl-CoA binding pore in the Acetyl-CoA structure. e Overlay of the cytosolic portion of the acetyl-CoA binding pore in the Acetyl-CoA structure (green), Apo_trans structure (brown) and the Apo_ground structure (black). f Overlay of monomers of the Acetyl-CoA structure (light green) and the Apo_ground structure (gray) shows a significant conformation change of TM1. g Overlay of the dimers of the Acetyl-CoA structure (green/light green), Apo_trans structure (brown/light brown), and the Apo_ground structure (black/gray) shows a large rotation movement. TM1 is hide for clarity. h–j Dimer interface of the Acetyl-CoA structure (h), the Apo_trans structure (i), and the Apo_ground structure (j). Note, all ligand(s) bound structures exhibit similar conformation as the Acetyl-CoA structure (Supplementary Figs. 6, 7b). For simplicity, Acetyl-CoA is not shown in the Acetyl-CoA structure except in panel d and e.

Fig. 4. Model for the transmembrane acetylation reaction catalyzed by HGSNAT.

a Structural model of the reaction cycle of HGSNAT (steps 1–6). TMDs are colored in blue and orange while the ECDs and TM1s are colored in magenta and pink. The acetyl-CoA binding pore is represented by its outline. HS (GlcN) is shown as a green hexagon while acetyl-CoA is represented by a yellow triangle (acetyl group) connected with a red oval (CoA). HGSNAT exhibits an equilibrium between the ground state and transition state without substrate bound (step 1) and transitions into the priming state when acetyl-CoA binds (step 2). HS binding (step 3) completes the catalytic ternary complex and allows the reaction to proceed (step 4). Subsequent release of both substrates (steps 5 and 6) allows HGSNAT to return to the equilibrium between ground and transition states. b Zoom in of the acetyl-CoA binding pore and the active site with critical residues shown in black stick and acetyl-CoA shown in cyan ball-and-stick. M282, F310, and F313, where conformational changes that gate the acetyl-CoA binding pore are observed, are highlighted in red. For simplicity, the reaction in only one monomer is illustrated.

Fig. 5. MPS IIIC variants mapped on the structure of HGSNAT.

a MPS IIIC variants mapped on HGSNAT. ECD is colored in light blue and the TM1 is in light pink. TMD is colored in wheat while the ECL loops are colored in light yellow. b Zoom in of the mutation hotspot 1 where substrates binding is affected. c Zoom in on the mutation hotspot 2 which influences the acetyl-CoA accessibility. d Zoom in of the mutation hotspot 3 at the interface between TMD and ECL.