Excited protein states of human tear lipocalin for low- and high-affinity ligand binding revealed by functional AB loop motion

Abstract

Human tear lipocalin (TL), a prominent member of lipocalin family, exhibits functional and structural promiscuity. The plasticity of loop regions modulates entry to the ligand pocket at the "open" end of the eight-stranded β-barrel. Site directed multi-distance measurements using fluorescence resonance energy transfer between functional loops register two excited protein states for low-and high-affinity ligand binding. At low pH, the longest loop AB adopts the conformation of the lowaffinity excited protein state that matches the crystal structure of holo-TL at pH 8. A "crankshaft" like movement is detected for the loop AB in a low pH transition. At pH 7.3 the holo-protein assumes a high-affinity excited protein state, in which the loop AB is more compact (RMS= 3.1Å). In the apo-holo transition, the reporter Trp 28 moves about 4.5 Å that reflects a decrease in distance between Glu27 and Lys108. This interaction fixes the loop AB conformation for the high-affinity mode. No such of movement is detected at low pH, where Glu27 is protonated. Data strongly indicate that the protonation state of Glu27 modulates the conformation of the loop AB for high-and low-affinity binding.

Probing Tertiary Structure of Proteins Using Single Trp Mutations with Circular Dichroism at Low Temperature

“…Unlike other lipocalins, TL shows significant conformational changes in the apo- to holo transition. 25 , 26 , 48 , 49 Accordingly, the position 124 shows marked displacement (4.90 Å) in the α-helix upon ligand binding (Figure 9 ). This suggests that the N-terminus residue, W124, is conformationally mobile to sample multiple conformations and is reflected in greater CD augmentation compared to other sites of the α-helix.…”

Ligand binding complexes in lipocalins: Underestimation of the stoichiometry parameter (n)

“…7, Table 2). Recombinant tear lipocalin has been taken as a surrogate for the apo-form without exposure to solvents for delipidation [19,55]. During expression tear lipocalin binds some lipids, but fewer than native tear lipocalin [56,57].…”

Cation-π Interactions in Lipocalins: Structural and Functional Implications

“…The conformation of Trp28 is driven by protonation states of the neighboring residues and is stabilized by ligand binding. Moreover, the distance measurements using FRET method have indicated that in the pH induced transition, Trp28 (originally Phe28) was moved, 4.5 Å, toward the loop EF (14). This result obtained in solution is concord with the crystal structures of TL in apo- and holo-forms (15, 30).…”

“…Human tear lipocalin (TL), also known as von Ebner's gland protein, is an archetypical ligand binding member of the lipocalin family (11). The enhanced promiscuity of TL for lipids is derived from structural plasticity and a large mouth as determined by site-directed tryptophan fluorescence (SDTF) (12–14). X-ray crystallography of TL is concordant with the SDTF derived solution structure (15).…”

“…Therefore, molecular mechanisms that impact ligand binding are functionally relevant. Progress has been made on the specific mechanistic features of ligand binding to TL (12–14, 24–30). In the binding cavity, fatty acids are predominantly oriented with the hydrocarbon tail buried in the cavity and the carboxyl group at the calyx mouth (29, 31).…”