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.

Figure 1

Positions of the residues that are considered for mapping the binding site of TL. Gray circles show locations of the C_α atoms of the residues. The side chains of all residues, but 35 and 115, are oriented inside the cavity. Single and double letters denote the identities of the β-strands and loop, respectively. The arrow shows the ligand entrance site of the cavity. The ribbon diagram of TL was generated from PDB 1XKI (20). The missing loop parts were built by DeepView/Swiss-PdbViewer v.3.7 (GlaxoSmithKline R&D) in accord with the structural data of tear lipocalin (18, 20).

Figure 2

Far-UV CD spectra of TL mutants. CD spectra were recorded using protein concentrations of 1.2 mg/mL in 10 mM sodium phosphate at pH 7.3. To reduce overlap, the spectra were shifted vertically. For comparison, the spectrum of wild-type TL (dashed line) was added to each spectrum of the mutant protein.

Figure 3

Near-UV CD spectra of TL mutants. The experimental conditions were the same as in Figure 2.

Figure 4

Binding curves of the fluorescence probes ANS (A), 1NPN (B), and TNS (C) to apoTL (5 μM). Emission λ values for ANS, 1NPN, and TNS binding were 465, 416, and 432 nm, respectively.

Figure 5

ANS binding to the mutants L115W, K114W, and K114C. Emission λ values in ANS binding experiments with L115W, K114W, and K114C were 462, 475, and 475 nm, respectively. Concentrations of the proteins were in the range of 5–6 μM.

Figure 6

Fluorescence spectra of E34W at various ANS concentrations. The numbers in the figure represent the ANS concentration (μM). The fluorescence spectra have been corrected for the inner filter effect.

Figure 7

Quenching the steady-state fluorescence of single Trp mutants by ANS. The fluorescence intensities were measured at the wavelengths of the respective emission maxima. Emission λ values in the experiments with L105W, C101W, M39W, and E34W were 348, 337, 332, and 344 nm, respectively. The excitation λ was 295 nm. The solid curves are the best fit for one binding site model.

Figure 8

Time-resolved fluorescence decay of single Trp mutants (A) L105W and (B) E34W with and without ANS. Solid curves are the best fit for a double exponential decay. Emission λ values in the experiments with L105W and E34W were 348 and 344 nm, respectively. The excitation λ was 295 nm.

Figure 9

Evidence for RET between Trp105 and bound ANS in the L105W–ANS complex. (A) Key: dotted line, corrected excitation spectrum (emission at 500 nm) of ANS (1.5 μM) in ethanol; dashed line, corrected excitation spectrum (emission at 500 nm) of the W17Y (5.0 μM)–ANS (2.5μM) complex; solid line, corrected excitation spectrum (emission at 500 nm) of the L105W (4.4 μM)–ANS (2.5 μM) complex. (B) Key: dashed line, excitation spectrum (emission at 348 nm) of L105W (4.4 μM) without ANS; solid line, difference excitation spectrum at an emission of 500 nm [spectrum of L105W–ANS – 0.67(spectrum of the W17Y–ANS complex shown in (A))]. The arrow indicates the position of the 0–0 band of the ¹L_b electronic absorption transition of Trp.

Figure 10

Evidence for RET between Trp101 and bound ANS in the C101W–ANS complex. (A) Key: dashed line, corrected excitation spectrum (emission at 500 nm) of the W17Y (5.0 μM)–ANS (2.5μM) complex; solid line, corrected excitation spectrum (emission at 500 nm) of the C101W (5.0 μM)–ANS (2.5 μM) complex. (B) Key: dashed line, excitation spectrum (emission at 337 nm) of C101W (5.0 μM) without ANS; solid line, difference excitation spectrum at an emission of 500 nm [spectrum of C101W–ANS – 0.63(spectrum of the W17Y–ANS complex shown in (A))].

Figure 11

Arrangement of amino acid residues, considered for ANS binding, in the crystal structure of TL (PDB 1XKI (18)). Residue Leu105 is modeled from the solution structure of TL by SDTF (20). Gray, blue, red, and yellow balls represent carbon, nitrogen, oxygen, and sulfur atoms, respectively. The numbers represent distances between the atoms (Å). The image was generated by ViewerLite 5.0 (Accelrys Inc.).

Figure 12

Docking solution for interaction of ANS with TL. The backbone of TL (PDB 1XKI) is shown in cyan in a ribbon representation. Gray, blue, red, and yellow sticks represent carbon, nitrogen, oxygen, and sulfur atoms, respectively. The position of ANS (energy level –11.0 kcal/mol) is that in best agreement with the experimental data. Distances, indicated with green dotted arrows, between the sulfonate group of ANS and the side chains of Glu34, His84, and Lys114 are 2.5, 2.4, and 3.0 Å, respectively. The image was generated by DeepView/Swiss-PdbViewer v.3.7 (GlaxoSmithKline R&D).

Figure 13

Displacement of DAUDA from the DAUDA(2 μM)–TL(4 μM) complex by various ligands at pH 7.3. Excitation and emission wavelengths were 345 and 498 nm, respectively. IC₅₀ values for each ligand are shown in parentheses. Solid curves are generated by fitting of the experimental data to one binding site model.