Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins

Abstract

Zinc is an essential nutrient in the body; it is required for the catalytic activity of many hundreds of human enzymes and virtually all biological processes, therefore its homeostasis and trafficking is of crucial interest. Serum albumin is the major carrier of Zn²⁺ in the blood and is required for its systemic distribution. Here we present the first crystal structures of human serum albumin (HSA) and equine serum albumin (ESA) in complex with Zn²⁺. The structures allow unambiguous identification of the major zinc binding site on these two albumins, as well as several further, weaker zinc binding sites. The major site in both HSA and ESA has tetrahedral geometry and comprises three protein ligands from the sidechains of His67, His247 and Asp249 and a water molecule. Isothermal titration calorimetric studies of a HSA H67A mutant confirm this to be the highest affinity Zn²⁺ site. Furthermore, analysis of Zn²⁺ binding to HSA and ESA proved the presence of secondary sites with 20-50-fold weaker affinities, which may become of importance under particular physiological conditions. Both calorimetry and crystallography suggest that ESA possesses an additional site compared to HSA, involving Glu153, His157 and His288. The His157 residue is replaced by Phe in HSA, incapable of metal coordination. Collectively, these findings are critical to our understanding of the role serum albumin plays in circulatory Zn²⁺ handling and cellular delivery.

Fig. 1. Crystal structure of ESA in complex with Zn²⁺. All structures of ESA–Zn²⁺ presented herein are superposed and a single Zn²⁺ for each site is shown as a representative. The overall fold in the ; 5IIH structure is shown as representative. Helices are represented by ribbons and Zn²⁺ by red spheres. Zn²⁺ binding sites are marked with numbers I–XVIII as in Table 3.

Fig. 2. The all-atoms superposition of residues within 8 Å from Zn²⁺ in site a of HSA–Zn²⁺ complex (; 5IJF, blue), and corresponding residues in (a) native HSA structure (; 1AO6, white) and (b) ESA–Zn²⁺ structure (2.5 mM Zn²⁺, pH 7.4 – ; 5IIH, green). Zinc ions are shown in grey, oxygen in red, sulfur in yellow, nitrogen in dark blue, coordination bonds with Zn²⁺ ion are marked with black dashed lines. Residue numbers refer to HSA.

Fig. 3. Representative structures showing dynamic behavior of His247 in ESA–Zn²⁺ complexes. (a) ESA–Zn²⁺, 2.5 mM Zn²⁺, pH 7.4; ; 5IIH. (b) ESA–Zn²⁺, 30 mM Zn²⁺, pH 7.4; ; 5IJE. (c) ESA–Zn²⁺, 15 mM Zn²⁺, pH 6.5; ; 5IIX. Residues are shown in sticks representation, zinc ion in grey, oxygen in red, nitrogen in dark blue, carbon in green. Coordination bonds are marked with black dashed lines. Grey grid represents 2mf_o – df_c map (σ – 1.0), orange – anomalous map (σ – 3.0).

Fig. 4. Secondary Zn²⁺ binding sites in the structures of ESA. Sites are numbered as in Table 3; only those coordinated by at least two protein residues are shown. Residues are shown in sticks representation, zinc ion in grey, oxygen in red, nitrogen in dark blue, carbon in green. Coordination bonds are marked with black dashed lines. Grey grid represents 2mf_o – df_c map (σ – 1.0), orange – anomalous map (σ – 3.0). Residue numbers refer to HSA; in case of non-conserved residues, the HSA amino acid is given in parentheses.

Fig. 5. Measurement of Zn²⁺ binding affinity to HSA, H67A HSA mutant, and ESA by ITC. (a) ITC data for Zn²⁺ binding to HSA and the H67A mutant form. (b) ITC data for Zn²⁺ binding to ESA. In all cases the respective protein (50 μm) was titrated with 5 μl injections of a 1.5 mM ZnCl₂ solution for 55 injections.