Ligand Binding Site of Tear Lipocalin:  Contribution of a Trigonal Cluster of Charged Residues Probed by 8-Anilino-1-naphthalenesulfonic Acid

Abstract

Human tear lipocalin (TL) exhibits diverse functions, most of which are linked to ligand binding. To map the binding site of TL for some amphiphilic ligands, we capitalized on the hydrophobic and hydrophilic properties of 8-anilino-1-naphthalenesulfonic acid (ANS). In single Trp mutants, resonance energy transfer from Trp to ANS indicates that the naphthalene group of ANS is proximate to Leu105 in the cavity. Binding energies of TL to ANS and its analogues reveal contributions from electrostatic interactions. The sulfonate group of ANS interacts strongly with the nonconserved intracavitary residue Lys114 and less with neighboring residues His84 and Glu34. This trigonal cluster of residues may play a role in the ligand recognition site for some negatively charged ligands. Because many drugs possess sulfonate groups, the trigonal cluster-sulfonate interaction can also be exploited as a lipocalin-based drug delivery mechanism. The binding of lauric acid and its analogues shows that fatty acids assume heterogeneous orientations in the cavity of TL. Predominantly, the hydrocarbon tail is buried in the cavity of TL and the carboxyl group is oriented toward the mouth. However, TL can also interact, albeit relatively weakly, with fatty acids oriented in the opposite direction. As the major lipid binding protein of tears, the ability to accommodate fatty acids in two opposing orientations may have functional implications for TL. At the aqueous-lipid interface, fatty acids whose carboxyl groups are positioned toward the aqueous phase are available for interaction with TL that could augment stability of the tear film.

A Simple Model-Free Method for Direct Assessment of Fluorescent Ligand Binding by Linear Spectral Summation

“…ANS binding to native TL as well as to many of its numerous mutants has been studied previously [34,25,23]. ANS binds within the cavity of TL [23] that results in an enhancement of the fluorescence intensity and a blue shift in fluorescence λ max (Fig.…”

“…ANS binds within the cavity of TL [23] that results in an enhancement of the fluorescence intensity and a blue shift in fluorescence λ max (Fig. 1).…”

Intracavitary Ligand Distribution in Tear Lipocalin by Site-Directed Tryptophan Fluorescence

“…Commonly, a unique solution for docking studies is presumed. When the docking studies yield multiple solutions for a ligand with similar binding energy, the result that better fits experimental data is chosen to represent the protein–ligand complex (for example, in TL ( 40 ), in β -lactoglobulin ( 41 )). Interestingly, although interaction energies have not been reported, retinoic acid could be docked to TL in two orientations ( 17 ).…”

Tear film lipids

“…First of all, the authors assumed that the major source of PL in tears was meibum. Second, Dean and Glasgow suggested that most of PL are bound to lipocalin (or tear specific pre-albumin) – a protein that had been previously shown to bind a range of compounds of lipid and non-lipid nature (Gasymov et al, 2008; Gasymov et al, 2001; Glasgow et al, 2010), including PL (Gasymov et al, 2005). Third they suggested, rather contradictorily considering the previous point, that the entire PC pool found in tears may form a monolayer of PC, thus fulfilling the goal proposed in the earlier work of McCulley and Shine (McCulley and Shine, 1997).…”