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Structural and biochemical insight into a modular β-1,4-galactan synthase in plants

Nature Plants volume 9, pages 486–500 (2023)Cite this article

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Abstract

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1) transfers galactose and arabinose to either extend or cap the β-1,4-galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal ___domain that represents the founding member of a new family of carbohydrate-binding module, CBM95, and a C-terminal glycosyltransferase family 92 (GT92) catalytic ___domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a ‘handshake’ orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.

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Fig. 1: The role of GalS1 in RGI synthesis.
Fig. 2: The structure of GalS1 obtained by X-ray crystallography.
Fig. 3: The CBM95 ___domain is the founding member of a new CAZy family.
Fig. 4: Conserved pattern positions that distinguish the GT92 family.
Fig. 5: Insights from docking and molecular dynamics simulations.
Fig. 6: Proposed mechanism of galactan synthesis by GalS1.

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Data availability

The diffraction data and crystallographic models that support the findings of this study are available at the Protein Data Bank (https://www.rcsb.org/) under PDB accession codes 8D3T and 8D3Z for GalS1 in the apo form and the GalS1 bound to Mn2+, respectively. The SAXS data and model have been deposited and are available from the SIMPLE SAXS (https://simplescattering.com) database under the accession code XSMHXTBH. Other data that support the findings of this study and any computer code used herein are available from the corresponding author upon request. Source data are provided as part of this paper.

Code availability

The software used for analysis of the crystallographic data is freely available online or from the authors of each software package. Any computer code used herein is available from the corresponding author upon request.

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Acknowledgements

Funding to support B.R.U., P.K.P., V.S.B. and Y.J.B. was provided by the Center for Bioenergy Innovation (CBI), from the US Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. The work conducted by H.V.S., P.D.A., J.H.P. and M.H. at the Joint BioEnergy Institute is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under contract no. DE-AC02-05CH11231 between LBNL and the US Department of Energy. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231. The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical Institute. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169. Funding for the SIBYLS beamline at the Advanced Light Source was provided in part by the Offices of Science and Biological and Environmental Research, US Department of Energy, under Contract DE-AC02-05BH11231 and NIGMS grant P30 GM124169-01, ALS-ENABLE. Funding for N.K. and R.T. was provided by R35 GM139656 and R01 GM130915. Work by K.W.M., J.-Y.Y. and D.C. was supported by the US National Institutes of Health grants R01 GM130915 and P41GM103390 (to K.W.M.).

Author information

Author notes

  1. These authors contributed equally: Pradeep Kumar Prabhakar, Jose Henrique Pereira.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA

    Pradeep Kumar Prabhakar, Rahil Taujale, Kelley W. Moremen, Natarajan Kannan & Breeanna R. Urbanowicz

  2. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA

    Pradeep Kumar Prabhakar, Digantkumar Chapla, Jeong-Yeh Yang, Kelley W. Moremen & Breeanna R. Urbanowicz

  3. Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oakridge, TN, USA

    Pradeep Kumar Prabhakar & Breeanna R. Urbanowicz

  4. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jose Henrique Pereira, Michal Hammel & Paul D. Adams

  5. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jose Henrique Pereira, Wanchen Shao, Paul D. Adams & Henrik V. Scheller

  6. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Wanchen Shao & Henrik V. Scheller

  7. Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA

    Vivek S. Bharadwaj

  8. Biosciences Center, National Renewable Energy Laboratory, Golden, CO, USA

    Yannick J. Bomble

  9. Department of Bioengineering, University of California, Berkeley, CA, USA

    Paul D. Adams

  10. Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA

    Henrik V. Scheller

Authors
  1. Pradeep Kumar Prabhakar
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  2. Jose Henrique Pereira
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  3. Rahil Taujale
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  4. Wanchen Shao
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  5. Vivek S. Bharadwaj
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  6. Digantkumar Chapla
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  7. Jeong-Yeh Yang
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  8. Yannick J. Bomble
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  9. Kelley W. Moremen
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  10. Natarajan Kannan
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  11. Michal Hammel
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  12. Paul D. Adams
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  13. Henrik V. Scheller
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  14. Breeanna R. Urbanowicz
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Contributions

P.K.P., J.H.P., R.T., W.S., V.S.B., N.K., M.H., P.D.A., H.V.S., D.C., J.-Y.Y. and B.R.U. designed and performed experiments, and analysed data. R.T., V.S.B. and N.K. performed computational simulations and machine learning studies. D.C. and J.-Y.Y. expressed proteins. K.W.M. and Y.J.B. designed experiments, analysed and interpreted data, and edited the paper. P.K.P., J.H.P., R.T., V.S.B., N.K., M.H., P.D.A., H.V.S. and B.R.U. wrote the paper. P.D.A., H.V.S. and B.R.U. conceived the project and B.R.U. led the project.

Corresponding author

Correspondence to Breeanna R. Urbanowicz.

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Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Neighbor-Joining consensus tree of GT92 peptide sequences from plants.

All full-length protein sequences were downloaded from Phytozome v1295 and generated using the software suite Geneious 2019.1. The position of PtGalS1 in the tree is highlighted with a gold star (☆). Plant species are abbreviated as follows: Selaginella moellendorffii (Sm), Solanum lycopersicum (Solyc), Populus trichocarpa (Potri), Physcomitrella patens (Pp), Panicum virgatum (Pavir), Oryza sativa (Os), Glycine max (Glymax), Eucalyptus grandis (EucGr), Daucus carota (DCAR), Citrus sinensis (Csi), Citrus clementina (Ciclev), Carica papaya (Cpap), Brachypodium distachyon (Bradi), and Arabidopsis thaliana (At).

Extended Data Fig. 2

Schematics of the native Populus trichocarpa GalS1 protein (gene loci Potri.005G258900) and the recombinant proteins used in the study.

Extended Data Fig. 3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SEC-MALS-SAXS analysis of GalS1, CBM95 and their variants.

SDS page of a, purified GalS1 WT protein and mutant variants. b, purified GalS1-CBM and its variants. 5 µg of protein was loaded in each well. See Extended Data Fig. 2 for construct information. Expression levels of soluble secreted sfGFP fusion proteins in the culture media were monitored by GFP fluorescence, indicated in Extended Data Tables S2 and S3. Each transfection experiment for mutant variants was performed at least once and two different SDS PAGE gels monitoring protein purification were generated. Data presented here are representative of the final purified product used in respective experiments. c, SEC-MALS-SAXS analysis of GalS1. SEC elution profile for the GalS1, along with masses calculated from MALS and radius of gyrations calculated from SAXS-frame collected across the SEC-elution peak. The masses confirm that untagged GalS1 is a dimer in solution (MW SAXS = 110 kDa, MW MALS = 119 kDa). Original uncropped images are provided in the Source Data.

Extended Data Fig. 4 Electron-density map of GalS1.

a, Stereo view of a sample of 2 |Fo | - |Fc | Фcalc electron density. b, Stereo view of Cα trace of the GalS1 as dimer.

Extended Data Fig. 5 Structural alignment of GalS1 with mammalian β-GlcNAc β-1,4-galactosyltransferases (β4GalTs).

The core catalytic ___domain of GalS1 (ivory) is well aligned with other galactosyltransferases (differently colored). GalS1 shows additional N- and C-terminal domains that are hypothesized to be necessary for binding the RG-I backbone to facilitate galactan chain elongation. GalS1 is aligned with a, Bos taurus Btβ4GalT1 (RMSD 4.7 over 104 residues, green);53 b, Homo sapiens Hsβ4GalT7 (RSMD 5.6 over 128 residues, magenta);35 c, Homo sapiens Hsβ4GalT1 (RMSD 4.2 over 104 residues, aqua);54 and d, Drosophila melanogaster Dmβ4GalT7 and (RMSD 5.7 over 136 residues, red)35.

Extended Data Fig. 6 Role of the conserved stem region on oligomerization and activity of GalS1.

Stem region interaction across monomers in dimeric GalS1. b, Dim plot 2D interaction map of residues that interact between stem regions in GalS1 homodimer. c, Comparison of GalT activity and d, AraT activity in WT and GalS1ΔSTEM variant. The values shown are mean values (bar) ± standard deviation (error bars) of a representative experiment for n = 3 technical replicates (red circles) and plotted using GraphPad Prism 9.5.0. e, Change in oligomerization state of GalS1ΔSTEM as compared to GalS1WT determined by SEC-MALS. f, Web logo showing conservation of residues beyond residue 96 in the predicted stem domains of plant GT92 proteins from Salix suchowensis, Populus alba, Populus trichocarpa, Jatropha curcas, Hevea brasiliensis, Ricinus communis, Manihot esculenta, Olea europaea, Carica papaya, Herrania umbratica, Cephalotus follicularis, Eucalyptus grandis, Cucurbita moschata, Camellia sinensis, Mangifera indica, Punica granatum, Pistacia vera, Durio zibethinus, Telopea speciosissima, Vitis riparia, Gossypium hirsutum, Quercus lobata, Quercus suber, Thalictrum thalictroides, Syzygium oleosum, Sesamum indicum, Gossypium australe, Morus notabilis, and Vitis vinifera. The sequence number on the X-axis refers to native GalS1 from Populus trichocarpa and Y-axis is in bits with error bars that indicate an approximate Bayesian 95% confidence interval. Thirty-three (n = 33) species of plants were examined in this experiment.

Extended Data Fig. 7 GT92 family conserved residues in GalS1 predicted to contribute to ligand specificity.

a, Highlighted residues studied in the current work. b, Hypervariable regions (HV), predicted to impart acceptor specificity to GalS1, and core-hydrophobic regions are shown in orange and yellow, respectively.

Extended Data Fig. 8 Donor specificity of GalS1 and β-1,4-galactotetraose acceptor dissociation constants (KD) of GalS1 WT and its variants.

a, Sugar-nucleotide specificity of GalS1. Reactions were carried out using 0.1 mM of UDP-substrate with 4 µg of the GalS1 enzyme in 50 mM HEPES, pH 7.5 in the absence of acceptor substrate. The values shown are mean values (bar) ± standard deviation (error bars) of a representative experiment for n = 3 technical replicates (red circles) and plotted using GraphPad Prism 9.5.0. b, Comparison of dissociation constants (KD) of PtGalS1 WT and its variants predicted for β-1,4-galactotetraose acceptor binding by microscale thermophoresis (MST). The values shown are mean values (filled circles) of the KD obtained after using KD fit model in the MO.Affinity Analysis software (NanoTemper Technologies) ± KD confidence (error bars). Error Bars represent standard deviation confidence (SD) values defined by the range where the KD falls with 68% of certainty, each point represents the mean of six sets of measurements (n = 6).

Extended Data Fig. 9 Comparison of galactosyltransferase activity of GalS1 WT and its variants by polysaccharide analysis using carbohydrate gel electrophoresis (PACE).

The results are representative of a single experiment.

Extended Data Fig. 10 Structural similarity between the experientially determined and AlphaFold2 predicted structures of GalS1.

a, Comparison of the X-ray structure of Populus trichocarpa GalS1 (97-495 residues; current study) determined in this work (in magenta) and the computationally predicted AlphaFold2 structure downloaded from the AlphaFold Protein Structure Database (AlphaFold id O22807;96,97 in green) of Arabidopsis thaliana GalS1 (97-496 residues). The root-mean-square deviation (RMSD; using Cα) of the aligned structures is 0.610 Å. b, Comparison of the X-ray structure of Populus trichocarpa GalS1 (97-495 residues; current study) determined in this work (in magenta) with the predicted AlphaFold2 structure of Populus trichocarpa GalS1 (97-495 residues) carried out in house using ColabFold98 (blue). The root-mean-square deviation (RMSD; using Cα) of the aligned structures is 0.593 Å.

Supplementary information

Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Source Data Extended Data Fig. 3

Unprocessed, uncropped SDS–PAGE gels.

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Prabhakar, P.K., Pereira, J.H., Taujale, R. et al. Structural and biochemical insight into a modular β-1,4-galactan synthase in plants. Nat. Plants 9, 486–500 (2023). https://doi.org/10.1038/s41477-023-01358-4

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