Table 1.
Crystal data and model refinement statistics for the structure of Can f 6.
Fig 1.
The overall architecture of Can f 6 displays the classic lipocalin structure.
(A) Eight antiparallel β-strands (yellow) form a central ligand binding cavity (βA-I) flanked by C-terminal α-helix (posteriorly located in the figure) and three 310-helices (red), with the connecting loops (L, in green) numbered. A disulfide bond (DS, cyan) clamps the C-terminal loop to the beta core via βD. The cavity opening is covered by L1 and a loop-310 helix-loop that links together βA and βD. Three core lipocalin Structurally Conserved Regions (SCR, purple) that hold the overall fold together are labeled: SCR1 connects the N-terminal loop to the C-terminal SCR3 which also interacts with SCR2. (B) Figure A is rotated by 90o out of the plane and shows the main residues of the core SCR. In SCR3, the side chain and distal guanidium group of a conserved Arg125 are stabilized by hydrogen bonds (dashed lines) to the backbone carbonyls of other SCR residues and a Cation-Pi interaction with a conserved SCR1 aromatic residue, here Trp21. Other residues in the SCR also form numerous non-covalent interactions. (C) Purified Can f 6 was assayed by Thermofluor shift to demonstrate the stability of our recombinant Can f 6. The average Tm is 67.9°C with standard deviation of 1.7°C.
Fig 2.
Primary structure alignment of lipocalins.
(A)The secondary structure for Can f 6 is drawn above the sequences. The colors correspond to the model of Can f 6 depicted in Fig 1 (A). Despite their conserved lipocalin architecture there is low sequence similarity between the dog lipocalins Can f 1, 2, 4, and Can f 6. The only disulfide bond (C, in red) occurs between C67 and C160. (B) Primary sequence alignment of the Can f 6 dog lipocalin, cat (Fel d 4), horse (Equ c 1), and mouse (Mus m 1). There is much more sequence similarity in these lipocalins compared to those of the dog lipocalins. Alignment residues are as: “*” Identical amino acid, “:” Similar amino acid, and “.” Slightly similar amino acid[35]. NxS/T (Blue N in blue boxes) are predicted N glycosylation sites (28), S/T (purple in black boxes) are predicted O glycosylation sites (29), S/T/T (orange) are predicted phosphorylation sites (30), and Y are predicted sulfation sites (31).
Fig 3.
The sequence alignment of lipocalins threaded onto the structure of Can f 6.
The sequence homology between the dog lipocalin Can f 6, 4, and 2 are more distinct compared to that between Can f 6 and Equ c 1, Fel d 4, and Mus m 1.
Fig 4.
Structural overlay of Can f 6 (yellow) on Can f 2 (red) and Can f 4 (grey) Equ c 1 (purple), and Mus m 1 (brown). The overall architecture of lipocalins is highly conserved with a β-barrel that encloses a hydrophobic core. The major differences are outside the core.
Fig 5.
Comparison of predicted post translational modification sites.
(A) Summary of glycosylation sites. (B) Three N-linked glycosylation residues (blue spheres) are predicted at Asn35, 52, and 75 in Can f 6; one in Can f 2 at Asn27; and two in Equ c 1 at Asn37 and 68. All these sites are on loops. The O-linked glycosylation site predicted in Can f 6 (purple spheres) is at the end of the C-terminus (Ser166) and is not modeled because this region of the structure is disordered. (C) Predicted phosphorylation sites (orange spheres). Phosphorylated Tyr22 and Ser18 in human tear lipocalin are present and labeled in Can f 6.
Table 2.
Molecular masses, numbers of positively and negatively charged amino acids and theoretical pI of Canf 6 and select lipocalins using ProtParam.
Fig 6.
A comparison of the ligand binding cavities in the different lipocalins.
Cavity residues and volume maps (red) were calculated using a rolling probe of 1.4Å and displayed as Connolly surfaces (25, 26, 27). (A) The overall ligand core is centrally located within the beta barrel and closed at the top by the L1 and L5 loops. (B) Residues from multiple strands of the β-barrel contribute to the ligand cavity core with hydrophobic amino acids and aromatic side chains in the interior of the ligand binding surface. (C) The ligand cavity volumes are fairly similar except for Equ c 1. (D) The ligand cavities of Can f 2 (red), Can f 4 (grey), Equ c 1 (purple), and Mus m 1 (brown) have a variable shape. Can f 6 and Equ c 1 have similar ligand cavity shape, with the slight differences between them likely reflects slightly differently shaped ligands.
Table 3.
Ligand binding pocket analysis of Can f 6 with the 17 main amino acid residues that define the shape of ligand binding cavity compared to homologous proteins.
Fig 7.
Comparison of the cavity capping residues of lipocalins.
In Can f 6, at the entrance of the calyx cavity His87 in L5 stabilizes L1 over the ligand binding mouth. Arg41 and Asp91 stabilizes His87 and the L1 loop and are highly conserved indicating a common capping mechanism between lipocalins. The His in Can f 6 is unique compared to a highly conserved Tyr that is observed in the other lipocalins reflecting a unique ligand binding mechanism. Volume map is portrayed in red as Connolly surfaces (26).
Fig 8.
Highly pure recombinant Can f 6 binds human serum IgE in individuals who have been tested for sensitization to unfractionated whole dander IgE.
Three of 5 samples from dog dander positive individuals have marked elevation in IgE binding to Can f 6. Mean and standard deviation of OD405nm for patients with dog IgE<0.35: 12.8±8.34 and IgE>5: 108±54.2 for Can f 6. Experiments were completed twice with the trend remaining the same. Overall p-value = 0.1199.
Table 4.
Demographics of human serum samples.