Fig 1.
(A) Multiple sequence alignment of CBM1 domains found in E. huxleyi, with benchmark CBM1 from Cel7A from T. reesei in the middle. The consensus sequence is visualized underneath the MSA. Residues are colored according to Zappo color scheme, with conserved residues colored more intensely. The three key aromatic residues involved in cellulose binding (position 7, 35, 38) are indicated with yellow triangles above the sequences. Alignment made with MUSCLE and formatted with Jalview 2 [32,33]. (B) Distance identity matrix of CBM1 domains found in E. huxleyi and benchmark CBM1 from Cel7A, displaying pairwise sequence identities in percentages (X of Y positions identical). The intensity of the blue color correlates with increasing sequence similarity. (C-D) Homology models of the (C) EHUX2 and (D) EHUX4 CBM1 domains, displayed as blue ribbons, with Cel7A-CBM1 as overlay, displayed in red. Cysteine bridges and the side-chains of residues that are important for binding are shown. Homology models were created by SWISS-MODEL [34] with the crystal structure of Cel7A-CBM1 (PDB ID: 1CBH [16]) as a template.
Fig 2.
Multiple sequence alignment of the investigated E. huxleyi CBM1 domains and selected related sequences.
In this MSA, the six E. huxleyi CBM1 domains are shown together with predicted non-fungal CBM1 domains, a selection of biochemically characterized CBMs from the CAZy database: CBM5 and fungal CBM1. The consensus sequence is visualized underneath the MSA. Residues are colored according to Zappo color scheme, with conserved residues colored more intensely. The three key aromatic residues involved in cellulose binding (position 7, 35, 38) are indicated with yellow triangles above the sequences. Alignment made with MUSCLE and formatted with Jalview 2 [32,33].
Fig 3.
Binding isotherms of all measured CBM1 proteins (A) with cellulose nanofibrils (CNF), (B) with chitin nanocrystals (ChNC) and (C) with bacterial cellulose (BC) as a ligand, displayed as μmol of bound AP-CBM1 protein / gram of ligand, against μmol of free protein.
Fig 4.
Binding of CBM1s in buffers with NaCl and CaCl2.
The effect of increasing ionic strength as well as calcium concentration was studied in detail for all proteins that showed binding to the ligands. The binding is displayed in percentage of AP-CBM1 protein bound / gram of carbohydrate ligand, in buffers containing increasing concentrations of NaCl or CaCl2. The CBM1s that show binding are accompanied by viewing guide lines. (A) Cel7A-CBM1 (B) EHUX2 (C) EHUX4(D) EHUX1b, EHUX3, EHUX5 with ChNC as binding ligand. Calcium concentration effect on binding to ChNC (E) and BC (F) for all proteins.
Fig 5.
Cel7A-EHUX1b segment shuffle experiment.
(A) Bar graph of the binding of Cel7A-CBM1, EHUX1b and 4 shuffled proteins, with the percentage of the protein bound to CNF, BC and ChNC shown on the y-axis. The shuffled proteins are labeled with four letter codes, where each of the four fragments is represented by the starting letter of the CBM1 they originate from (C = Cel7A; E = EHUX1b). (B) Sequences of Cel7A-CBM1, EHUX1b and the shuffled proteins. The four segments from Cel7A-CBM1 are color coded, corresponding with the 3D structure of Cel7A-CBM1 shown in (C). (C) Cel7A-CBM1 crystal structures with the segments use to create the shuffled proteins in four different colors. 3D structure shown as cartoon, with a view on the binding face as well as a front-side view. Image created with Chimera [40], PDB ID: 1CBH [16].