Architecture of the two metal-binding sites in prolactin

Abstract

Metal binding by members of the growth hormone (GH) family of hematopoietic cytokines has been a subject of considerable interest. However, beyond appreciation of its role in reversible packing of GH proteins in secretory granules, the molecular mechanisms of metal binding and granule formation remain poorly understood. Here, we investigate metal binding by a GH family member prolactin (PRL) using paramagnetic metal titration and chelation experiments. Cu²⁺-mediated paramagnetic relaxation enhancement measurements identified two partial metal-binding sites on the opposite faces of PRL composed of residues H30/H180 and E93/H97, respectively. Coordination of metal ions by these two sites causes formation of inter-molecular bridges between the PRL protomers and enables formation of reversible higher aggregates. These findings in vitro suggest a model for reversible packaging of PRL in secretory granules. The proposed mechanism of metal-promoted PRL aggregation lends insight and support to the previously suggested role of metal coordination in secretory granule formation by GH proteins.

Figure 1

(A) Alignment of the hGH structure (pink; PDB: 1BP3) in complex with zinc (gray sphere) and the free hPRL structure (blue; PDB: 1RW5). (B) Sequence alignment for hGH, hPL, and hPRL regions that include residues involved in Zn²⁺ coordination is shown.To see this figure in color, go online.

Figure 2

(A) Cu²⁺ binding does not induce conformational change in hPRL. Overlay of ¹⁵N-HSQC spectra of free hPRL (red) and hPRL:Cu²⁺ 1:1.9 (blue) is shown. (B) Examples of titration curves are shown: dependence of peak intensity loss with added Cu²⁺. (C and D) Per-residue ¹⁵N Γ₁ measured for hPRL at different conditions is shown. Shaded areas show the position of the four α-helices in the hPRL sequence. Dashed lines show values equal to two standard deviations of the corresponding distribution. The error bars show errors of Γ1 calculated as a sum of errors of paramagnetic and diamagnetic R1. (C) 100 μM hPRL:400 μM Cu²⁺ is shown: (1:4) sample (blue) at pH 7. The data shown in yellow were recorded after addition of 800 μM EDTA to the sample. (D) 200 μM hPRL at different concentrations of Cu²⁺ is shown: 100 μM (1:0.5; blue) and 400 μM (1:2; yellow) at pH 7. (E) Comparison of hPRL PREs measured at different pH is shown. Blue: 100 μM Cu²⁺:200 μM hPRL (1:0.5) at pH 7 is shown; yellow: 30 μM Cu²⁺:100 μM hPRL (1:0.3) at pH 5.5 is shown. To see this figure in color, go online.

Figure 3

Two metal-binding sites on the hPRL surface. (A) The structure of hPRL (PDB: 1RW5) is shown. Colored spheres show the residues affected by metal binding. Orange color represents residues that are sensitive to metal binding at pH 7.0 and 5.5, and yellow indicates residues that show high PRE for only one pH condition. (B) H30 and H180 coordinate the metal ion in MBS1. An ensemble of 20 hPRL structures calculated using MoSART with restraints between Cu²⁺ (orange) and residues of MBS1 is shown. (C) MBS2 by itself is not sufficient to tightly bind a metal ion. The additional pair of ligands provided by another PRL molecule creates a fully liganded MBS. An ensemble of 20 hPRL structures calculated by MoSART with restraints between Cu²⁺ (orange) and residues of MBS2 is shown. To see this figure in color, go online.

Figure 4

Metal-induced aggregation of 25 μM WT-hPRL (orange), MBS1-hPRL (blue), and MBS2-hPRL (green) determined by ultraviolet absorption at 280 nm. Absorption at pH 7.0 is shown by solid lines and at pH 5.5 by dashed lines. Each data point is an average of duplicate measurements and the error bars correspond to the difference. To see this figure in color, go online.

Figure 5

Models for hGH and hPRL aggregation. (A) The position of the H27, H30, and H180 MBS compared with the position of the E93 and H97 MBS on hPRL is shown. The MBSs are depicted in red. (B) A model for metal-mediated aggregation in hPRL is shown. The availability of two metal coordination sites on opposite sides of hPRL can result in large, amorphous, metal-mediated aggregates. (C) Position of the MBS and the four glutamic acids in GH (depicted in red) that possibly act as coordinating residues for intramolecular metal bridging is shown. (D) A possible mechanism for the metal-mediated dimerization of hGH is shown. In contrast to hPRL, the metal-binding sites in hGH abrogate the formation of higher aggregates by occluding the two sites at the dimer interface. To see this figure in color, go online.