Conserved positions in the two expansin domains
Standard numbering schemes for the N- and the C-terminal expansin domains (from positions 11 to 105 and 114 to 186, respectively) were applied to identify conserved positions (≥70% occurrence) in the superfamilies ‘Bacterial expansins’, ‘Fungal expansins’, and ‘Plant expansins’ (Table 2 ). In both expansin domains, glycine was the most frequently conserved amino acid (Tables S3 andS4 ). In the N-terminal expansin domain, nine positions were conserved in the three superfamilies (‘Bacterial expansins’, ‘Fungal expansins’, and ‘Plant expansins’): threonine 12, glycine 21, alanine 36, glycine 53, proline 74, aspartate 82, leucine 83, phenylalanine 88, and glycine 97 (Table S3 ). In the superfamily ‘Bacterial expansins’, further seven positions were highly conserved (≥90% occurrence): valine 58, glycine 60, glycine 63, aspartate 71, serine 84, alanine 87, and isoleucine 91; in the superfamily ‘Fungal expansins’, additional highly conserved amino acids are cysteine 23, phenylalanine 25, tryptophan 44, cysteine 52, cysteine 55, methionine 68, and leucine 81; and in the superfamily ‘Plant expansins’ highly conserved amino acids are alanine 22, cysteine 23, glycine 24, glycine 49, cysteine 52, cysteine 55, cysteine 60, cysteine 61.8, threonine 70, and phenylalanine 81.
Due to our conservation analysis, five of the previously proposed six cysteines14 were highly conserved in the superfamily ‘Plant expansins’, three conserved cysteines were found in the superfamily ‘Fungal expansins’, and none in the superfamily ‘Bacterial expansins’ (Table 2 and https://doi.org/10.18419/darus-735). The conserved cysteines at standard positions C23 and C52, C55 and a cysteine upstream of the N-terminal expansin domain standard numbering, and C60 and 61.8 were proposed to form disulfide bonds14.
In the C-terminal expansin domain, only two positions are conserved in the three superfamilies ‘Bacterial expansins’, ‘Fungal expansins’, and ‘Plant expansins’: tryptophan 149 and glycine 179 (Table S4 ). In addition, in the superfamily ‘Bacterial expansins’, highly conserved positions are lysine 119, glycine 121, tryptophan 126, proline 137, tyrosine 157, asparagine 158, glycine 166, threonine 175, and aspartate 176; in the superfamily ‘Fungal expansins’, glycine 121, serine 123, tryptophan 126, phenylalanine 127, glutamine 130, valine 131, asparagine 133, valine 143, serine 143.1, aspartate 146, arginine 154, tyrosine 157, asparagine 158, phenylalanine 160, glycine 164, valine 172, and threonine 175; and in the superfamily ‘Plant expansins’ highly conserved amino acids are glycine 136, glycine 157, and tryptophan 160. From the conserved positions, superfamily-specific motifs were derived. In the N-terminal domain of ‘Bacterial expansins’, these motifs are VpGP (standard positions 58-61, “p” is the abbreviation for polar amino acids selected from 40) and HLDL (80-83) (Table 3 ); in the superfamily ‘Fungal expansins’ the motifs T(F/W)YG (12-14 and 14.1), GTAnS (34-38, “n” is the abbreviation for non-polar amino acids selected from40), VpGn (58-61), and HLDL (80-83) were identified; and in the superfamily ‘Plant expansins’, the motifs T(F/W)YG (12-14 and 14.1) in EXPA and EXPB, GGACGYG (20-26) in minor modifications in all four plant expansin groups, and HFDL (80-83) in EXPA and EXPB were identified (Tables 3 , S3 and https://doi.org/10.18419/darus-735). In the C-terminal expansin domain of the superfamily ‘Bacterial expansins’, the motif QVRNH (130-134) was conserved; in the superfamily ‘Fungal expansins’ the motifs QVnN (130-133), LEVSTDGD (141-146, including 143.1 and 143.2 as insertions relative to the reference sequence), GGG (164-166), and VDVRVT (170-175) were identified. At the standard positions 170 to 175, the motif LSFpVT is included in sequences of EXPA. Sequences from the superfamilies ‘Bacterial expansins’ and ‘Fungal expansins’ were found to share the motif, KpG(S/T)S (119-123); and pGS exists also in EXPB. EXLA and EXLB were found to share the motif, YLA (126-128). The motif WGA exists in minor modifications in all four groups of ‘Plant expansins’ (Tables 3 , S4 and https://doi.org/10.18419/darus-735).