Structural basis for matriglycan synthesis by the LARGE1 dual glycosyltransferase

Figures

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  Fig 1. LARGE1 dimerizes in solution. (a) A schematic diagram for the domain organization in the LARGE1 protein. (b) Purification of the recombinant LARGE1 proteins. Chromatograms from SEC for constructs with (LARGE1ecto, upper image) or without (LARGE1ΔCC, lower image) the CC domain. Insets show Coomassie-stained SDS-PAGE for the purified proteins. (c) Mass-photometry analysis of the two LARGE1 proteins at a concentration of 30 nM. The fraction of counts is noted for the two main peaks in the data sets that corresponds to the monomeric (~80 kDa) and dimeric (~160 kDa) forms of LARGE1. The rest of the counts (6% and 5%, for with and without CC, respectively) do not belong to these two main peaks. These are representative measurements that were repeated multiple times using different protein batches. (d) 2D class averages of LARGE1 particles from the two forms. For each LARGE1 form, two separate classes are shown, representing two distinct and perpendicular views. The 2-fold symmetry axes are shown as dashed yellow lines (for in-plane axes) or as an oval shape (for out-of-plane axis). Same-type domains are marked with asterisks.

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  Fig 2. Single particle cryo-EM maps of LARGE1. Reconstructed maps of the LARGE1ecto (upper part) and LARGE1ΔCC (lower part). The gold standard FSC curves are shown on the left and density maps colored by the local resolution estimates are shown on the right. One half map that was used as a molecular replacement model is highlighted with a dashed rectangle.

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  Fig 3. The dimeric structure of LARGE1. (a) Ribbon representation of the two LARGE1 monomers that make the asymmetric unit of the crystal. The Xyl-T, Linker region, and GlcA-T regions are highlighted in pink, hot pink, and gray on one monomer, respectively. The N- and C- termini are noted. Upper inset shows a 2Fo-Fc electron density map of the Xyl-T active site at the indicated sigma level. Lower inset shows a superimposition of the two modeled LARGE1 chains. (b) A fit of the LARGE1 dimer as appeared in the asymmetric unit of the crystal into the EM density map of the LARGE1ecto. (c) An individual fit of two LARGE1 monomers into the EM density map of the LARGE1ΔCC. (d) Analysis of the dimer interface. Upper image shows a LARGE1 monomer in a surface representation. The Xyl-T, Linker region, and GlcA-T regions are highlighted in pink, hot pink, and gray, respectively. Fully buried surface residues due to dimerization are shown in orange (probe size of 1.4 Å). Partially buried surface residues (i.e., making cavities) due to dimerization are shown in yellow (probe size of 2.0 Å). Two patches of buried surfaces on the Xyl-T and GlcA-T domains are marked with dashed oval lines. Lower image…

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  Fig 4. The Xyl-T domain of LARGE1. (a) Superimposition of the Xyl-T domain from LARGE1 (pink) with XXYLT1 (PDB: 4wn2, green). Inset shows a closeup view of the active site. The conserved residues that coordinate the Mn2+ are noted, as well as a UDP and a growing substrate that were resolved in the structure of XXYLT1. (b) Surface representation of the LARGE1 Xyl-T domain. The aspartic acids of the DXD motif are colored yellow. The UDP and the growing substrate derived from the structure of XXYLT1 are shown as sticks.

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  Fig 5. The GlcA-T domain of LARGE1. (a) Superimposition of the GlcA-T domain from LARGE1 (grey) with K4CP (PDB: 2z86, green). Inset shows a closeup view of the active site. Mn2+ atoms of LARGE1 (purple) and of K4CP (green) are shown as semi-transparent spheres. A UDP from the K4CP structure is shown in purple. The conserved DXD motif and histidine residues that participate in the coordination of the Mn2+ are noted. (b) The GlcA-T active sites of the two LARGE1 monomers (left and right) in the crystallographic asymmetric unit are shown with an Fo-Fc difference map (green mesh, σ = 5) calculated after omitting the Mn2+ atoms. (c) Surface representation of the LARGE1 GlcA-T domain. The aspartic acids of the DXD motif are colored yellow. The UDP derived from the structure of K4CP is shown as sticks.

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  Fig 6. Matriglycan synthesis by the LARGE1 dimer. The closest active sites of the Xyl-T and GlcA-T domains are located of different monomers. The LARGE1 dimer is shown using a surface representation and the catalytic domains are colored as in Fig 3a. The two LARGE1 monomers are highlighted by different tones. A yellow arrow shows the openings of the closest Xyl-T and GlcA-T catalytic sites.

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  Fig 7. Disease causing mutations in LARGE1. (a) Mapping of four mutations on the structure of LARGE1. Insets show closeup views of the native residues at the mutation sites. (b) Western blot analysis using anti-His antibody showing the expression of soluble LARGE1-His WT or E509K mutant. Samples include the total cell lysates and cells’ media as indicated. (c) Similar to ‘b’, a western blot analysis showing the cells’ media and lysates from cell transfected with LARGE1-WT, -S331F, -C443Y, or -W495R. Lysate from non-transfected cells is shown as a negative control (NC).

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  Fig 8. Putative electrostatic interaction of LARGE1 with matriglycan. Surface electrostatic potentials of the LARGE1 dimer from the Xyl-T (top) and from the GlcA-T (bottom) sides of the dimer. Blue to red colors indicate 5 kT/e to -5 kT/e, respectively.