A molecular mechanism for modulating plasma Zn speciation by fatty acids

Abstract

Albumin transports both fatty acids and zinc in plasma. Competitive binding studied by isothermal titration calorimetry revealed that physiologically relevant levels of fatty acids modulate the Zn-binding capacity of albumin, with far-reaching implications for biological zinc speciation. The molecular mechanism for this effect is likely due to a large conformational change elicited by fatty acid binding to a high-affinity interdomain site that disrupts at least one Zn site. Albumin may be a molecular device to "translate" certain aspects of the organismal energy state into global zinc signals.

Figure 1

Domain structure of albumin and fatty acid binding sites. Overlaid structures with PDB codes: 1bj5, HSA with five myristates, pink (the protein backbone is also shown); 1e7e, HSA with 10 decanoates, green; 1gnj, HSA with seven arachidonates, light-yellow. FA1–5, major sites; fa6–10, minor sites.

Figure 2

Binding events at the domain I/II interface affect several His Hε1 resonances. (A) Interface with the disrupted Zn-binding site A (magenta) and interdomain His residues likely to be affected by fatty acid binding in site FA2. The 11 resolved carbons of Myr2 in PDB entry 1bj5 are shown as pink spheres. (B) Effects of 1 molar equiv of Zn and 5 molar equiv of OCT on His Hε1 resonances. Peaks 1 and 4 are assigned to His67 and His247; full titration data are shown in Figure S3.

Figure 3

Competitive binding of Zn and OCT to BSA studied by ITC. (A) Zn binding to BSA (25 μM) in the presence and absence of 5 molar equiv of OCT. (B) OCT binding to BSA and Zn₁BSA. Open circles with blue fits correspond to binary systems and filled circles with red fits to ternary systems. In each case, 34 injections of 8 μL of 333 μM ligand (Zn or OCT) were delivered over 16 s with 240 s between injections for complete equilibration.

Figure 4

Different binding modes for (A) medium- and (B) short-chain fatty acids in site FA2 on HSA. Fatty acid molecules are shown in pink. The colored surfaces represent Analytical Connolly surfaces of the residues forming the binding pocket. In both models, the carboxylate headgroup interacts with R257, and the hydrophobic half-pocket in domain II (blue) is formed by residues L250, L251, A254, A258, L283, and L284. (A) HSA with bound MYR, based on PDB entry 1bj5. Three C atoms have been added to the C11 chain resolved in the X-ray structure. Domain I (orange and yellow) contributes to the fatty acid binding site an extended half-pocket comprising residues R10, L14, F19, L22, V23, A26, L66, and Y150. The complete pocket can be formed only if the zinc site (labeled residues) is disrupted. (B) HSA with OCT and Zn²⁺ (purple) bound simultaneously. OCT is short enough to be accommodated predominantly in the domain II pocket. Hydrophobic residues L14, F19, L22, and L155 form a new half-pocket without disrupting the zinc site.

Figure 5

Competitive binding of metals and MYR to BSA. (A) Effect of increasing amounts of MYR on the zinc-binding capacity of BSA. ITC curves for titrations of 333 μM Zn²⁺ into 25 μM BSA in the presence and absence of varying amounts (0–5 molar equiv) of MYR in 50 mM Tris/50 mM NaCl (pH 7.2). The fits (Figure S8) allowed estimates of the ratio of site A availability, as shown in (B). A clear downward trend was observed. A 4:1 MYR:Zn molar ratio suppressed occupation of site A almost completely. A second binding site was also affected by fatty acid binding (see D). (C) ITC curves for titrations of 500 μM MYR into 12.5 μM BSA or Zn₁BSA. These titrations were carried out in H₂O because of the insufficient solubility of MYR in Tris buffer. The fits (Figure S9) correspond to a model with one set of binding sites to estimate the stoichiometry for the highest-affinity sites. This equaled 5.0 ± 0.3, and the average log K was 6.3 ± 0.4 in the absence and 5.9 ± 0.4 in the presence of Zn²⁺. More complex fits were possible, but the data were insufficient to justify them. (D) ¹¹¹Cd NMR spectra of Cd₂BSA recorded in the absence and presence of 5 molar equiv of MYR. Peaks A and B were both suppressed by MYR.