Abstract
Purpose: :
Reduction of the conserved disulfide bond, (Cys61-Cys153), of human tear lipocalin (TL) produces conformational relaxation and permits a swap of retinol for native lipids (Biochemistry 1998, 37, 2215-2225) by an unknown mechanism. Solution and crystal structure data suggest that ligand binding is influenced by the motion of the C-D hairpin in TL. The hypothesis that a redox switch modulates ligand binding kinetics through C-D hairpin motion was investigated.
Methods: :
Disulfide-less TL mutants, C101L/C61S, C101L/C153S and C101L/C61S/C153S were constructed and compared to apo-TL with far and near UV circular dichroism. Time dependent ligand binding was performed by adding TL or mutant S61S153 to NBDC12-HPC (4 µM) or NBD cholesterol (2 µM) and measuring steady state fluorescence ex= 410 nm, em= 527 nm. Cys81 of TL mutant W62C81 was fluorescently labeled (83% efficiency) with 1,5-IAEDANS. Fluorescence resonance energy transfer between the Trp residue (donor) and the labeled 1,5 ANS group (acceptor) was measured using time-resolved intensity decay data from a phase/modulation multi-frequency domain fluorometer. Distances between the residues 62 and 81 (Trp62-1,5ANS81) in both disulfide reduced and intact forms were calculated.
Results: :
Near UV circular dichroism spectra showed decreased signal intensity but unaltered secondary structure with disulfide elimination, results similar to disulfide reduction. Upon disulfide removal, time constants of ligand binding for the NBD-cholesterol- TL complex decreased from 12 to 3 seconds, compared to those of NBD phospholipid-TL from 33 to 22 seconds. Fluorescence resonance energy transfer experiments reveal the distance between residues 62 and 81 closes with disulfide reduction from 29.6 to 27.6 Å (apo-form) and 26.2 to 24.3 Å (holo-form).
Conclusions: :
Disulfide-thiol exchange in TL results in enhanced flexibility and faster equilibrium in ligand binding. Conformational changes coincide with C-D hairpin motion. The process affects binding kinetics in a ligand specific manner. A bulky cholesterol derivative, a low affinity ligand to TL with the disulfide intact, shows greater enhancement of binding with disulfide reduction compared to a less bulky ligand. The results show the redox switch alters selective ligand kinetics to trigger the ligand swap in TL. Since the disulfide bond in TL is reduced by thioredoxin, an enzyme reportedly elevated in tears during inflammation, ligand specificity may be altered by variation in environmental conditions.
Keywords: protein structure/function • lipids • lacrimal gland