Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity
Introduction
Tear lipocalin (TL) is secreted by the lacrimal glands and comprises 15–33% of the protein content of tears [1], [2], [3], [4]. The identical protein is secreted by Von Ebner’s gland and has been identified in the prostate and skin [5], [6], [7]. TL is the principal lipid binding protein in tears and binds a broad array of lipid molecules including cholesterol, fatty acids, glycolipids and phospholipids [8]. TL, acting as a lipid carrier in tears, could potentially protect the eye in several ways. TL may scavenge lipid from the corneal surface and prevent dry spots from forming on the cornea. Solubilization of lipid in tears promotes optical clarity. Lipids bound to TL in the aqueous portion of tears provide a reservoir of lipid molecules that upon release, could migrate to the film surface to reduce water evaporation and inhibit microbial infection of the exposed ocular surface [8]. Several other putative functions have been identified for TL including antimicrobial activity [9], cysteine proteinase inhibition [10], and transport of sapid molecules in saliva [5], as well as retinol in tears [6]. The relative binding affinity of TL for its native ligands has not been previously studied and would clarify its potential function as a transporter of specific lipids in the tear film.
Members of the lipocalin family share certain common biologic activity and structural features. Most of the lipocalins acts as transporters of small insoluble lipids. X-Ray crystallography of several lipocalins have revealed eight β strands arranged in a calyx-shaped configuration with a hydrophobic binding pocket [11], [12], [13]. Although the crystal structure for tear lipocalin has not been resolved, studies of the solution structure of TL reveal many features in common to the other lipocalins. The amount of β structure, 31–53%, that has been directly observed in TL by circular dichroism (CD) is consistent with that of other lipocalins [14]. Site-directed tryptophan fluorescence has resolved the twisted β structure of the G strand in TL that is similar to other lipocalins [15]. The buried configuration of certain residues (e.g., F99) of the G strand in TL are comparable to residues (e.g., L103) of the G strand that exist in the hydrophobic cavity of β-lactoglobulin [15]. In addition, a disulfide motif in TL is conserved in the lipocalin family and plays a role in ligand affinity and contributes to protein rigidity [16]. TL and other lipocalins display similar conformational changes with ligand binding. For example, both TL and retinol binding protein exhibit increased aromatic side chain asymmetry and rigidity with ligand binding [17], [18]. Under acidic conditions both of these proteins transit through a molten globule state and release their ligands [17], [18].
The aromatic residues in TL include five tyrosine, three phenylalanine, and one tryptophan. Trp 17, in the A strand of TL, shows changes in dynamic exposure and side chain asymmetry that reflect conformational alterations with ligand binding and release [17]. The tryptophan at this position is the only amino acid residue that is invariably conserved throughout the lipocalin family [7]. It has been suggested that the analogous tryptophan in β-lactoglobulin, Trp 19, located at the tip of the calyx, may have a role in maintaining local structure and preventing oxidation of bound retinol [19]. Because TL contains only one tryptophan, an ideal opportunity was afforded to study the role the conserved Trp17 in maintaining structure and ligand binding by site directed mutagenesis.
Section snippets
Materials and methods
The fluorescent fatty acid analogs 11-(((5-(dimethlyamino)-1-naphthalenyl)sulfonyl)amino)undecanoic acid (DAUDA) and 16-(9-anthroyloxy)palmitic acid(16-AP) were obtained from Molecular Probes, Eugene, OR. Dansyl-dl-α-aminocaprylic acid (DACA), stearic acid, palmitic acid, lauric acid, myristol, tristearin, cholesteryl stearate, cholesterol, and l-α-lysophosphatidylcholine (purified from bovine liver and containing primarily palmitic and stearic acids) were obtained from Sigma, St. Louis, MO. A
Electron paramagnetic resonance spectroscopy
The composite EPR signals demonstrate the signals of free and bound spin-labeled compound with progressively increased concentration of ligand (Fig. 1A). It is evident that with low concentrations of spin label (Fig. 1A, a) the composite signal reflects relatively less free signal than at higher concentrations of C12 spin label (Fig. 1A, e). The binding of C12 spin label to both apo- and holo-TL can be plotted as saturation curves (Fig. 1B). The plots indicate that both apo and holo-TL achieve
Discussion
It is evident from the EPR data that apo- and holo-TL have a single binding site for the C-12 spin label. The diminished affinity demonstrated by holo-TL for the spin-labeled ligand reflects the fact that holo-TL carries an assortment of native ligands including fatty acids [8]. We surmise that the alteration in affinity occurs because the C-12 spin label, a lauric acid analog must displace the native ligands. These results indicated to us that the best characterization of ligand affinity for
Acknowledgements
We thank Joseph Horwitz for providing access to the Jasco 600 spectropolarimeter, and Wayne Hubbell for providing spin-labeled reagents and access to the Varian 109 spectrometer. This research was supported by USPHS NIH EY 11224, EY 00331 and an unrestricted grant from Research to Prevent Blindness.
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