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The typical F‐type lectin domain (FLD) has an L‐fucose‐binding motif [HX(26)RXDX(4)R/K] with conserved basic residues that mediate hydrogen bonding with alpha‐L‐fucose. About one‐third of the nonredundant FLD sequences in the publicly available databases are "atypical"; they have motifs with substitutions of these critical residues and/or variations in motif length. We addressed the question if atypical FLDs with substitutions of the critical residues retain lectin activity by performing site‐directed mutagenesis and assessing the glycan‐binding functions of typical and atypical FLDs. Site directed mutagenesis of an L‐fucose‐binding FLD from Streptosporangium roseum indicated that the critical His residue could be replaced by Ser and the second Arg by Lys without complete loss of lectin activity. Mutagenesis of His to other naturally substituting residues and mutagenesis of the first Arg to the naturally substituting residues, Lys, Ile, Ser, or Cys, resulted in loss of lectin activity. Glycan binding analysis and site‐directed mutagenesis of atypical FLDs from Actinomyces turicensis, and Saccharomonospora cyanea confirmed that Ser and Thr can assume the L‐fucose‐binding role of the critical His, and further suggested that the residue in this position is dispensable in certain FLDs. We identified, by sequence and structural analysis of atypical FLDs, a Glu residue in the complementarity determining region, CDR5 that compensates for a lack of the critical His or other appropriate polar residue in this position. We propose that FLDs lacking a typical FLD sequence motif might nevertheless retain lectin activity through the recruitment of other strategically positioned polar residues in the CDR loops. © 2018 IUBMB Life, 71(3):385–397, 2019 [ABSTRACT FROM AUTHOR]