The 1.8-Å Crystal Structure of Human Tear Lipocalin Reveals an Extended Branched Cavity with Capacity for Multiple Ligands

Abstract

In contrast with earlier assumptions, which classified human tear lipocalin (Tlc) as an outlier member of the lipocalin protein family, the 1.8-Å resolution crystal structure of the recombinant apoprotein confirms the typical eight-stranded antiparallel ␤-barrel architecture with an ␣-helix attached to it. The fold of Tlc most closely resembles the bovine dander allergen Bos d 2, a well characterized prototypic lipocalin, but also reveals similarity with ␤-lactoglobulin. However, compared with other lipocalin structures Tlc exhibits an extremely wide ligand pocket, whose entrance is formed by four partially disordered loops. The cavity deeply extends into the ␤-barrel structure, where it ends in two distinct lobes. This unusual structural feature explains the known promiscuity of Tlc for various ligands, with chemical structures ranging from lipids and retinoids to the macrocyclic antibiotic rifampin and even to microbial siderophores. Notably, earlier findings of biological activity as a thiol protease inhibitor have no correspondence in the three-dimensional structure of Tlc, rather it appears that its proteolytic fragments could be responsible for this phenomenon. Hence, the present structural analysis sheds new light on the ligand binding activity of this functionally obscure but abundant human lipocalin.

Molten globule state of tear lipocalin: ANS binding restores tertiary interactions

“…The residues 96-101 exhibit the alternating periodicity, which is characteristic of β-sheets. This feature is very consistent with the solution model and crystal structure of TL resolved by site-directed tryptophan fluorescence (SDTF) and x-ray crystallography, respectively [13,14].…”

“…The solution structure of TL was resolved by site directed tryptophan fluorescence and revealed a capacious cavity that confers promiscuity in ligand binding [13]. These findings were verified by crystallography of TL [14].…”

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

“…Trp has become a reliable chromophore (label) for studies of the structurefunction relationship of the proteins. The solution structure of human tear lipocalin (TL), has been resolved with site-directed tryptophan fluorescence (SDTF) [7] and, later, verified by xray crystallography [8]. Trp is also utilized extensively by other spectroscopic techniques, such as absorption and circular dichroism (CD) spectroscopies [9,10].…”

Evidence for internal and external binding sites on human tear lipocalin

“…Therefore, ion pair formation between a sulfonate group of ANS and an intracavitary positively charged side chain, can not be excluded, particularly in light of the fact that a positively charged residue Lys 114 is located inside the cavity [17,18]. However, several inferences can be drawn from these experiments.…”

“…Several putative functions have been assigned to TL such as scavenging lipid from the corneal surface to prevent the formation of lipid induced dry spots [13], solubilization of lipid in tears (2), antimicrobial activity [14], cysteine proteinase inhibition [15], endonuclease activity [16] and scavenging of toxic molecules [10]. The solution structure of TL has been resolved by site directed tryptophan fluorescence and verified by crystallography of TL [17,18]. A capacious cavity with short interstrand loops C-D and E-F at the entrance confers promiscuity in ligand binding [18] compared to other more selective lipocalins such as such as RBP.…”