Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.
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Raw EM videos and maps have been deposited at the Protein Data Bank and Electron Microscopy Data Bank under accession codes 7SP7 and EMD-25367; 7SP6 and EMD-25366; 7SP8 and EMD-25368; 7SP9 and EMD-25369; and 7SPA/EMD-25370 for the UDP-bound, D302N apo, UDP-GlcNAc-bound, primed (closed) and primed (open) states, respectively.
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We thank K. Dryden and M. Purdy from the MEMC at the University of Virginia as well as A. Wier and the support staff at the NCI-NCEF. E.P. and J.S. acknowledge the support of Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), and the Research Foundation–Flanders (FWO) for supporting the nanobody discovery and thank E. Beke for technical assistance. J.Z. and F.P.M. were supported by NIH grant R21AI148853. R.H. was supported by NIH grant R01GM101001 (awarded to J.Z.). R.A.C. and P.J.S. are supported by Wellcome (208361/Z/17/Z). P.J.S.’s laboratory is supported by awards from the BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1) and the MRC (MR/S009213/1). P.J.S. acknowledges the University of Warwick Scientific Computing Research Technology Platform for computational access.
The authors declare no competing interests.
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Extended data figures and tables
Comparison of HAS primary sequences from Chlorella virus (Cv), Homo sapiens (Hs) and Streptococcus equisimilis (Se). Topology predictions were performed using TopCons50. Cylinders indicate secondary structure elements observed in Cv-HAS.
Extended Data Fig. 2 Identification, data collection, and processing of Cv-HAS bound to two nanobodies and UDP.
(a) Increased melting temperature of Cv-HAS in the presence of Nb872. Protein melting was measured based on enzymatic activity detected by quantifying the release of UDP in real time. (b) HA biosynthesis in the presence of the indicated nanobodies and based on quantification of 3H-labelled HA by scintillation counting. Data is normalized relative to product yields in the absence of nanobodies. Error bars represent deviations from the means with n = 3 independent experiments. (c) Representative autoradiography of 14C-labelled HA produced in the presence of the indicated nanobodies. The experiment has been repeated at least 4 times with essentially identical results. NC: Negative control in the absence of UDP-GlcNAc substrate (for panel b) or UDP-GlcA (for panel c). PC: Positive control in the absence of nanobody. Lyase: Hyaluronan lyase treatment prior to SDS-PAGE. (d) This workflow produced the UDP-bound Cv-HAS structure.
Extended Data Fig. 3 Cryo-EM data collection and processing of Cv-HAS D302N in the presence of substrate.
This workflow generated the apo, substrate-bound, primed, and primed with open channel Cv-HAS structures.
(a-d) Map overview, estimated resolution based on FSC, and particle orientation distribution. (e) Secondary structure elements and topology of Cv-HAS. (f-j) TM helices 2 to 6 of Cv-HAS. (k) TMH3-4 extracellular loop. (l) The extracellular TMH5-6 loop. (m) The QxxRW motif. (n) The C-terminal cytosolic helix. (o) The unresolved TMH5-IF3 loop. All maps are contoured at 7.0σ.
(a) Relationship of evolutionarily coupled residues within Cv-HAS’ TM and GT regions, generated in MapPred based on 65,535 sequences. TMH1 is shown at its predicted location as a violet cylinder. (b) RoseTTAfold models of full-length Cv-HAS. Cv-HAS is shown as a surface and its TMH 2 as a blue cylinder. TMH1 is shown as a cartoon at its predicted locations. (c) An AlphaFold2 predicted structure of human HAS2 (coloured blue to red from its N- to C-terminus) overlaid with the Cv-HAS structure shown as a grey cartoon and semi-transparent surface. (d) TMH1 remains disordered when two cytosolic nanobodies are used for cryo-EM analyses. (e) Catalytic activity of TMH1 truncated Cv-HAS. Left: Western blot of IMVs used for in vitro activity measurements. Right: Catalytic activity of the indicated Cv-HAS mutants expressed relative to the wild type enzyme. The assay quantifies 3H-labelled HA by scintillation counting. Control reactions in the absence of UDP-GlcA served as background and are subtracted. Error bars represent deviations from the means with n = 3 independent experiments.
(a) Representative map regions for modelled lipids contoured at 7.0σ (from the UDP-GlcNAc bound set). (b) 2D slice from MD simulations of Cv-HAS (black area) within a POPE bilayer. Water and lipid densities are coloured blue and green, respectively. Right panel: Lipid contact times with selected channel residues. (c) Comparison of the Cv-HAS (rainbow coloured from the N- to C-terminus) and RsBcsA (grey, 4P00). Cellulose associated with BcsA is shown as black sticks. Helices are shown as cylinders except BcsA’s N-terminal two TMHs, which are shown as coils.
(a and b) Map quality for UDP-GlcNAc, UDP, and Mn2+ ligands. (c) Comparison of UDP and UDP-GlcNAc positions. (d) Map for the priming loop in nucleotide bound states. (e) Representative map for the GlcNAc primer. (f) Map for the priming loop in the primed states. (g) Contact point of TMH2 (open in blue, closed in grey) with IF1 in the primed state. (h and i) Map quality for TMH2 in a closed position (UDP-GlcNAc bound) and open position. All maps are contoured at 7.0σ.
Shown is an autoradiogram of 14C-labelled HA after SDS-PAGE. The experiment has been repeated at least 3 times with essentially identical results.
(a and b) Reaction schemes for UDP-GlcA and UDP-GlcNAc hydrolysis. (c and d) Raw absorbance measurements. (e) Quantification of hydrolysis rates in the presence of increasing monosaccharide concentrations. Blue and Red: Hydrolysis of UDP-GlcNAc and UDP-GlcA, respectively. Light and dark colours represent control reactions in the absence of enzyme. Right panel: Background subtracted hydrolysis rates. Error bars represent deviations from the means with n = 3 independent experiments.
Extended Data Fig. 10 Likely mechanism of alternating substrate polymerization and comparison with cellulose synthase.
(a) Superimposition of substrate-bound and primed Cv-HAS structures. The close distance between the primer and donor sugar is indicated by grey bars. (b) Contact likelihood between C231 and GlcNAc for the systems in a over the last 125 ns of each simulation. In the case of GlcNAc being in both donor and acceptor positions, both GlcNAc units are less likely to bind C231, and exhibit very high variance regarding binding poses. Of particular note, the chance of both GlcNAc being in the C231 pocket at the same time is very low (ca. 1.5 ± 0.8%). (c) Cv-HAS is superimposed with the Rhodobacter sphaeroides (Rs) BcsA-B complex (PDB: 4P00) based on secondary structure matching. Rs-BcsA-B is coloured grey and Cv-HAS is coloured blue and green for its TM and GT domains. The cellulose polymer associated with Rs-BcsA-B is shown as black sticks.
Supplementary Discussions 1–3, Supplementary Tables 1–3 and Supplementary References.
A continuous volume series representing density differences at the active site. Overlay of densities from a simple 3D variability display job corresponding to Fig. 2a. Trp342 is shown in sticks, UDP-GlcNAc and the GlcNAc primer are shown as ball and sticks coloured grey and cyan for carbon atoms, respectively.
Movement of the priming loop. Output from a simple 3D variability display overlayed with a Chimera morph created from models of the Primed open and the UDP-GlcNAc-bound states. The video oscillates from the primed open state through the UDP-GlcNAc-bound state and back to the primed open state.
Substrate-induced global conformational changes of Cv-HAS. Output from a simple 3D variability display overlayed with a Chimera morph created from models of the Primed open and the UDP-GlcNAc bound states. Oscillation from the primed open state through the UDP-GlcNAc-bound state and back to the primed open state.
Tilting of the TMH2 and opening of the putative HA channel. Output from a simple 3D variability display overlayed with a Chimera morph created from models of the Primed open and the primed closed states. Blue, TMH2; orange, TMH4; green, TMH6. Oscillation from the primed closed state through the primed open state and back to the primed closed state.
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Maloney, F.P., Kuklewicz, J., Corey, R.A. et al. Structure, substrate recognition and initiation of hyaluronan synthase. Nature 604, 195–201 (2022). https://doi.org/10.1038/s41586-022-04534-2
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