Supplementary Material
Cellulose synthases: new insights from crystallography and modeling
Erin Slabaugh1*, Jonathan K. Davis1*, Candace H. Haigler1,2, Yaroslava G. Yingling3, Jochen Zimmer4
1Department of Crop Science, North Carolina State University, Raleigh, North Carolina, USA
2Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA
3Department of Materials Science and Engineering, North Carolina State University, Raleigh, North Carolina, USA
4Center for Membrane Biology, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA
Corresponding author: Davis, JK. ()
*These authors contributed equally to this manuscript
Table S1. Known missense mutations in plant CESAs with phenotypic descriptions and information about the surrounding secondary structure of mutations that occur within the cytosolic domainaAllele / Mutation / CESA / Mutated region in CESA / Phenotype in addition to decreased cellulose content / Distance (Å) from DCD motif / Mutation position / Refs
eli1-1 / S301F / AtCESA3 / TMH2 / Aberrant lignin deposition, stunted growth / [S1,S2]
ixr1-6 / S377F / AtCESA3 / Cytosolic domain / Reduced growth, isoxaben resistance / 12 / GTA beta sheet strand 1 / [S3]
fra6 / R362K / AtCESA8 / Cytosolic domain / Reduction of fiber cell thickness / 45 / Known mutation farthest from the active site in PCR region / [S4]
eli1-2 / A522V / AtCESA3 / Cytosolic domain / Aberrant lignin deposition, stunted growth / 18 / Substrate binding pocket; Appears to be on a flexible loop bordering the substrate binding pocket, but not directly adjacent to the active site / [S1,S2]
rsw1-1 / A549V / AtCESA1 / Cytosolic domain / Radial cell swelling and short root when grown at warmer temperature / 17 / Alpha helix that precedes GTA beta sheet strand 3, which contains DCD; separated from eli1-2 by a single alpha helix. / [S5,S6]
irx3-5 / D524N / AtCESA7 / Cytosolic domain / Partially collapsed xylem, reduced guard cell size, increased transpiration efficiency, compact rosette, darker leaves, cellulose content not determined / 0 / Active site (Mg binding), GTA beta sheet strand 3 / [S7]
fra5 / P557T / AtCESA7 / Cytosolic domain / Reduced fiber cell thickness, fragile fibers, weak stems, semi-dominant / 17 / GTA beta sheet strand 5; position homolgous to thantos and repp3 mutation / [S8]
repp3 / P578L / AtCESA3 / Cytosolic domain / Defective PIN1-HA localization, isoxaben resistance, swollen roots with ectopic lignification / 17 / GTA beta sheet strand 5; position is the same as than and homologous to fra5 mutation / [S9]
than / P578S / AtCESA3 / Cytosolic domain / Shorter primary roots and hypocotyls, radial cell swelling, semi-dominant / 17 / GTA beta sheet strand 5; position is the same as repp3 and homologous to fra5 mutation / [S10]
any1 / D604N / AtCESA1 / Cytosolic domain / Dwarfism, reduced anistropic growth, disordered cellulose microfibrils in epidermal cells of inflorescence stem, cellulose content not altered, decreased cellulose crystallinity, decreased velocity of CSCs at the plasma membrane, increased xylose, reduced elongation of root / 27 / Near a putative IF helix based on structural alignment to BcsA (Figure 1B) / [S11]
lycos / G620E / AtCESA1 / Cytosolic domain / Decreased cellulose crystallinity, reduced height, quinoxyphen resistance / 19 / C-terminus of predicted IF1 / [S3]
cev1 / G617E / AtCESA3 / Cytosolic domain / Decreased cellulose content in roots, cellulose content not altered in aerial tissue, increased production of jasmonate and ethylene, constitutive expression of stress response genes, stunted roots, thickened roots and hypocotyls / 10 / Internal GTA helix adjacent to beta strand 4 / [S12, S13]
rsw1-2 / G631S / AtCESA1 / Cytosolic domain / Radial swelling, aberrant embryogenesis, lethal / 11 / Internal GTA helix adjacent to beta strand 4 / [S14]
irx1-2 / S679L / AtCESA8 / Cytosolic domain / Collapsed xylem / 14 / Substrate binding pocket / [S15]
rsw1-45 / E779K / AtCESA1 / Cytosolic domain / Short seedlings with radially swollen cells at control temperature and warmer temperature, aberrant embryogenesis / 6 / Substrate binding pocket / [S16]
irx1-1 / D683N / AtCESA8 / Cytosolic domain / Collapsed xylem / 4 / Substrate binding pocket / [S17]
rsw1-20 / D780N / AtCESA1 / Cytosolic domain / Short seedlings with radially swollen cells at control temperature and warmer temperature, aberrant embryogenesis, no trichomes / 4 / Substrate binding pocket / [S16]
mur10-2 / H734Y / AtCESA7 / Cytosolic domain / Dwarfism, lower fucose and xylose content, higher arabinose content, cellulose content not determined / 15 / Helix that frames GTA beta sheet strands 3 and 4, histidine extends in the direction of strands 5 and 6 / [S18, S19]
lew2-2 / L792F / AtCESA8 / Between TMH 3/4 / Higher levels of osmolytes and ABA, increased drought and osmotic stress tolerance, transcripts of stress markers induced, cellulose content not determined / [S20]
ags1-2 / A903V / AtCESA1 / TMH4 / Decreased cellulose crystallinity, quinoxyphen resistance, increased CSC velocity at the plasma membrane, semi-dominant / [S21]
S1-60 / G905D / OsCESA9 / TMH5 / Brittle culm / [S22]
mre1/je5b / G916E / AtCESA3 / TMH5 / Sucrose induced root swelling, increased root growth in response to ethylene inhibitor AIB, smaller rosette leaves, defective PIN1-HA localization / [S9,S23]
bc11 / G858R / OsCESA4 / Between TMH5/6 / Brittle culm, dwarfism, increased xylose, galactose and arabinose, reduced thickness of sclerenchyma, reduced OsCESA4 at the plasma membrane / [S24]
ixr1-2 / T942I / AtCESA3 / Between TMH5/6 / Isoxaben resistance, decreased cellulose crystallinity, increased CSCs velocity at the plasma membrane / [S3,S21]
ixr1-1 / G998D / AtCESA3 / TMH7 / Isoxaben resistance, cellulose content not determined / [S25]
ixr2-1 / R1064W / AtCESA6 / TMH8 / Isoxaben resistance, semi-dominant, cellulose content not determined / [S26]
rsw5 / P1056S / AtCESA3 / C-term / Radial swelling, decreased cellulose content at warmer temperature / [S27]
aA list of all known missense mutations in plant CESAs organized by appearance in the protein sequence. For mutations that occur within the cytosolic domain, the GhCESA1 modeled structure was used to determine the distance (Å) of the mutation from the active site, designated as the first D in the DCD motif. Abbreviations for mutants are as follows: aegeus (ags), any1 (anisotropy) brittle culm (bc), constitutive expression of VSP1 (cev), ectopic lignin (eli), fragile fiber (fra), irregular xylem (irx), isoxaben resistant (ixr), Joanna EMS mutant (je5)b, leaf wilting (lew), multiple response expansion (mre), murus (mur), radial swelling (rsw), regulator of PIN (pin-formed) polarity (repp), thanatos (than).
bPersonal correspondence with Marie-Theres Hauser
Table S2. Putative glucan-interacting residues in AtCESA1, AtCESA8 and GhCESA1.aResidue in BcsA / AtCESA1 / AtCESA8 / GhCESA1 / Identical, conserved or semi-conserved
H276 / Q595 / Q504 / Q493 / :
M300 / V612 / V521 / V510 / :
F301 / F613 / F522 / F511 / *
W383 / W822 / W725 / W714 / *
N412 / N855 / N759 / N748 / *
F419 / T862 / T766 / T755
R423 / L866 / L780 / L759
F426 / Y869 / Y783 / Y762 / :
Y433 / C876 / C790 / C769
E439 / F882 / F796 / F775
Y455 / A903 / I817 / I796
S476 / E924 / E828 / E817 / .
E477 / Q925 / Q829 / Q818 / :
V551 / V1001 / L904 / L893 / :
V555 / Y1005 / Y908 / Y897
W558 / W1008 / W911 / W900 / *
aPutative glucan-interacting residues were identified in AtCESAs and GhCESA1 based on protein sequence alignment with BcsA (Figure 3, Figure S1). The putative glucan-interacting residues did not change compared to the full-length sequence alignment if the CSR and/or the P-CR insertions in plant CESA were omitted individually or together. However, some of the putative glucan-interacting residues in CESA were different depending on whether CLUSTALW or CLUSTAL OMEGA was used to make the sequence alignment. This discrepancy is likely due to the low amino acid conservation in this region, which is typical of TMHs in general. Both of these alignment algorithms identified the same putative glucan-interacting residues within TMH3 of CESA (analogous to TMH5 in BcsA).
Figure S1. Full-length protein sequence alignment of AtCESA1, 3, 4, 6, 7 and 8, GhCESA1 and BcsA (PilZ domain omitted). CLUSTALW was used to generate the alignment using full-length protein sequences from AtCESA1, 3, 4, 6, 7 and 8, GhCESA1 and a truncated sequence of BcsA with the regulatory PilZ domain removed. Gray shade indicates predicted TMHs, blue shade indicates BcsA IFs, carets (^) indicate putative glucan-interacting residues in BcsA as well as putatively orthologous glucan-interacting residues in CESA, arrowheads indicate Trp or Tyr that reside within a TMH and orange circles denote the locations of known missense mutations in AtCESAs.
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