Volume 3, Issue 12, Pages 1379-1393 (December 1995) Crystal structure of human Charcot–Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins  Demetrios D Leonidas, Boris L Elbert, Zeqi Zhou, Hakon Leffler, Steven J Ackerman, K.Ravi Acharya  Structure  Volume 3, Issue 12, Pages 1379-1393 (December 1995) DOI: 10.1016/S0969-2126(01)00275-1

Figure 1 . Stereoview of a portion of the final 1.8 å 2 | Fo | – | Fc | electron-density map for CLC protein. The map is contoured at 1.0σ level. (The figure was generated using the program O [68].) Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 2 . The three-dimensional structure of human CLC protein. (a) Orthogonal views of CLC protein structure. β strands are presented in cyan, the two small 310 helices in red and the loops in yellow. (b) Stereoview of the Cα backbone of CLC protein molecule. (The diagrams were drawn using MOLSCRIPT [74].) Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 2 . The three-dimensional structure of human CLC protein. (a) Orthogonal views of CLC protein structure. β strands are presented in cyan, the two small 310 helices in red and the loops in yellow. (b) Stereoview of the Cα backbone of CLC protein molecule. (The diagrams were drawn using MOLSCRIPT [74].) Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 3 . Structural details of CLC protein. (a) Structure-based sequence alignment of CLC protein, bovine heart galectin-1 (1SLC) and human galectin-2 (1HLC) as determined using the program SHP [72]. Every tenth residue in the CLC sequence is numbered. Residues that form the carbohydrate-binding site are boxed. The dots indicate gaps inserted for optimal alignment. In CLC protein the solvent inaccessible residues (less than 20 å2 of exposed surface as calculated using the program DSSP [71]) are indicated by # and residues involved in crystal packing contacts are marked by ∗. Polar interactions are indicated by lower case letters and water mediated interactions ≤4.0 å are highlighted in grey boxes. Secondary structural elements (β strands F1–F5, S1–S6b) as determined by DSSP [71] are also shown. Residues which are identical between CLC protein and galectins are highlighted in bold letters. (b) Diagram of hydrogen bonds involving main-chain atoms in the CLC protein structure. Squares indicate residues in β strands and circles indicate residues in 310 turns. Arrows represent hydrogen bonds. (Figure drawn using the program HERA [75].) (c) Stereoviews of the structural alignment of CLC protein (black) on bovine heart galectin-1 (1SLC, red), bovine spleen galectin-1 (1SLT, green) and human galectin-2 (1HLC, cyan). The high degree of structural conservation is apparent. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 3 . Structural details of CLC protein. (a) Structure-based sequence alignment of CLC protein, bovine heart galectin-1 (1SLC) and human galectin-2 (1HLC) as determined using the program SHP [72]. Every tenth residue in the CLC sequence is numbered. Residues that form the carbohydrate-binding site are boxed. The dots indicate gaps inserted for optimal alignment. In CLC protein the solvent inaccessible residues (less than 20 å2 of exposed surface as calculated using the program DSSP [71]) are indicated by # and residues involved in crystal packing contacts are marked by ∗. Polar interactions are indicated by lower case letters and water mediated interactions ≤4.0 å are highlighted in grey boxes. Secondary structural elements (β strands F1–F5, S1–S6b) as determined by DSSP [71] are also shown. Residues which are identical between CLC protein and galectins are highlighted in bold letters. (b) Diagram of hydrogen bonds involving main-chain atoms in the CLC protein structure. Squares indicate residues in β strands and circles indicate residues in 310 turns. Arrows represent hydrogen bonds. (Figure drawn using the program HERA [75].) (c) Stereoviews of the structural alignment of CLC protein (black) on bovine heart galectin-1 (1SLC, red), bovine spleen galectin-1 (1SLT, green) and human galectin-2 (1HLC, cyan). The high degree of structural conservation is apparent. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 3 . Structural details of CLC protein. (a) Structure-based sequence alignment of CLC protein, bovine heart galectin-1 (1SLC) and human galectin-2 (1HLC) as determined using the program SHP [72]. Every tenth residue in the CLC sequence is numbered. Residues that form the carbohydrate-binding site are boxed. The dots indicate gaps inserted for optimal alignment. In CLC protein the solvent inaccessible residues (less than 20 å2 of exposed surface as calculated using the program DSSP [71]) are indicated by # and residues involved in crystal packing contacts are marked by ∗. Polar interactions are indicated by lower case letters and water mediated interactions ≤4.0 å are highlighted in grey boxes. Secondary structural elements (β strands F1–F5, S1–S6b) as determined by DSSP [71] are also shown. Residues which are identical between CLC protein and galectins are highlighted in bold letters. (b) Diagram of hydrogen bonds involving main-chain atoms in the CLC protein structure. Squares indicate residues in β strands and circles indicate residues in 310 turns. Arrows represent hydrogen bonds. (Figure drawn using the program HERA [75].) (c) Stereoviews of the structural alignment of CLC protein (black) on bovine heart galectin-1 (1SLC, red), bovine spleen galectin-1 (1SLT, green) and human galectin-2 (1HLC, cyan). The high degree of structural conservation is apparent. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 4 . The lectin domain in CLC protein. This domain is compared with bovine spleen galectin-1 complexed with N-acetyl-lactosamine [21]; human galectin-2 complexed lactose [22]; coral tree lectin complexed with lactose [76]; β-D-glucanase complexed with 3,4-epoxy-butyl-β-D-cellobioside [45]; the N-terminal domain of asparagine amidase [44]; Human serum amyloid P component [48] and Wing-1 from Vibrio cholerae neuraminidase [50]. Bound metal ions are shown as green spheres. The N and C termini are shown as blue and red spheres, respectively. The coordinates of the lectin domains of the various proteins have been transformed after superposition onto CLC protein using the program SHP [72]. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 5 . Superposition of residues in CLC protein and bovine spleen galectin-1 in the vicinity of the carbohydrate-recognition site. The residue labels are from the CLC protein structure. The backbone is shown in cyan. The side chains of CLC protein are shown as coloured atoms and those of galectin-1 in brown. N-acetyl-lactosamine (LAC) from the galectin-1 structure is shown in green. Bound water molecules in the CLC structure at the carbohydrate-recognition domain are shown as cyan spheres. (The figure was generated using MOLSCRIPT [74].) Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 6 . Low-affinity binding of CLC protein to N-acetyl-D-glucosamine and lactose. CLC protein in lysates of AML14.3D10 cells showed a one to two fraction delay in elution, compared with the major protein peak, when the lysate was run on an (a) N-acetyl-D-glucosamine–agarose or (e) lactose–agarose column. (b) This weak binding could be inhibited by adding 150 mM N-acetyl-D-glucosamine to the lysis and column running buffer. (d) The presence of a non-competing sugar (D-mannose) in the lysis and running buffer did not affect binding (c). CLC did not bind to D-mannose agarose. (f) Wild-type recombinant CLC (wt-rCLC) from transiently transfected COS-7 cells showed the same one fraction delay in elution. (g) Mutation of the Cys29 and Cys57 residues to Gly (the double mutant) did not block the weak binding of rCLC to the lactose–agarose column. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 7 . Carbohydrate-binding activity of crystal-derived and affinity-purified CLC protein. Crystallized and resolubilized CLC from either (a) lyophilized or (b) freshly prepared crystals showed no binding activity to N-acetyl-D-glucosamine-agarose. Either BSA or horse myoglobin (1 mg) was mixed with the CLC protein as an elution marker before loading onto the column. Functionally active carbohydrate-binding CLC protein could be affinity chromatography purified (ACP) using 3.5 M MgCl2 for elution from the antibody column (d), but elution at high pH (triethylamine pH 11.5) completely inactivated the carbohydrate-binding activity of the purified protein (c), a lability shared by other galectins. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)

Figure 8 . A view of the residues in the potential LPLase active site in the CLC protein structure showing residues Tyr100, His114 and the water molecule form the potential catalytic triad, as observed in the native CLC protein structure. Amongst residues Asp98, Asp113 and Glu119, one residue may replace the water molecule in the formation of the catalytic triad in the active form of the structure. Structure 1995 3, 1379-1393DOI: (10.1016/S0969-2126(01)00275-1)