Millisecond timescale dynamics of human liver fatty acid binding protein: testing of its relevance to the ligand entry process

Abstract

For over a decade, scientists have been attempting to know more about the conformational dynamics of fatty acid binding proteins (FABPs), to answer the puzzling question of how ligands could access the internalized binding site(s). Conformational exchange of FABPs on the microsecond to millisecond timescales has been found in many FABPs and offers an important hypothesis for the ligand entry mechanism. Despite the potential significance, the validity of this hypothesis has not been verified yet. In this study, the slow dynamics of human liver fatty acid binding protein (hLFABP) that was shown previously to be highly flexible on millisecond timescales was quantitatively characterized in detail. In addition, the interaction between hLFABP and 1,8-ANS was studied using NMR spectroscopy, and the kinetic rate of ANS association to hLFABP was measured. We believe the current result excludes the possibility that the intrinsic millisecond dynamics of hLFABP represents a critical conformational reorganization process required for ligand entry, but implies that it may represent the exchange between the apo-state and a state resembling the singly-bound conformation. Furthermore, we suggest these results show that the ligand-entry related functional dynamics could occur on the microsecond/submicrosecond timescales, highly encouraging future computational studies on this topic.

Figure 1

hLFABP residues undergoing conformational exchange (green). Based on the backbone assignment of hLFABP (27), these residues are identified as Y9, Q10, L11, N16, I24, I31, G39, V40, E42, I43, F52, T53, I54, T55, A56, G57, V60, N63, E64, F65, V67, E74, M76, T77, V81, V94, T95, T96, K98, V103, T104, E105, T112, N113, M115, K123. The protein structure is displayed using UCSF Chimera (39).

Figure 2

Relaxation dispersion profiles of the residues with intrinsic conformational exchange. The residues that displayed R_ex ≥ 1.5 s⁻¹ at both 500 MHz and 800 MHz fields were fitted with the two-state exchange model, and the kinetics parameters (k₁ and k₋₁) and chemical shift difference (Δω) were extracted. Δω values are given in angular frequencies at 500 MHz field. The experimental data collected at 500 MHz and 800 MHz fields are represented as o and ^∗, respectively. The solid lines are theoretical curves. The errors in k₁, k₋₁, and Δω were estimated using the jackknife method, and they were similar to those estimated using the Monte Carlo method.

Figure 3

Representative residues in NMR titration experiments. The protein concentration was estimated to be ∼0.46 mM; the concentrations of ANS were: 0 (black), 0.11 (green), 0.23 (purple), 0.46 (red), 0.92 (orange), 1.8 (cyan), and 3.57 (light purple) mM. The direction of chemical shift walking due to ligand binding at the high (low) affinity site is indicated by the black (blue) arrow, (K59 did not show resonance shift on binding at the high affinity site); the minor change of the direction due to additional weak association is indicated by the dashed brown arrow.

Figure 4

Kinetics of hLFABP-ANS interaction. The hLFABP-ANS samples were prepared by adding ANS into the solution of hLFABP with final molar ratios (ANS/hLFABP) of (A) ∼1:4 and (B) ∼1:2, respectively. The experimental data of representative residues recorded at 500 MHz (o) and 800 MHz (^∗) fields were fitted with the two-state exchange model (solid lines) globally as described in the Materials and Methods.

Figure 5

The relaxation dispersion profiles of representative residues. The ^∗ represents the effective relaxation rates measured using the hLFABP sample, in which the low affinity site was ∼1/2 saturated. The o represents the effective relaxation rates measured using the apo-hLFABP sample. All the experiments were conducted using an 800 MHz Bruker machine at 20°C. The relaxation rates (^∗) were fitted with the theoretical curve for demonstration purpose, which should represent the millisecond exchange that can be effectively suppressed at ν_CP of 960 Hz. It should not be confused with the fast exchange of ligand association/dissociation at the low affinity site.

Figure 6

Histogram of CCSP of hLFABP on addition of ANS (at ∼1:1 ratio).

Figure 7

Comparison of protein regions showing resonance shift and those showing conformational exchange in the absence of ligands. Residues displaying significant resonance shift (CCSP ≥ 0.05 ppm) are shown in yellow and orange. Residues displaying significant R_ex (≥1.5 s⁻¹) are shown in yellow and green. The yellow region displayed both characteristics.