Probing the coordination environment of the human copper chaperone HAH1: characterization of Hg(II)-bridged homodimeric species in solution

Abstract

Although metal ion homeostasis in cells is often mediated through metallochaperones, there are opportunities for toxic metals to be sequestered through the existing transport apparatus. Proper trafficking of Cu(I) in human cells is partially achieved through complexation by HAH1, the human metallochaperone responsible for copper delivery to the Wilson and Menkes ATPase located in the trans-Golgi apparatus. In addition to binding copper, HAH1 strongly complexes Hg(II), with the X-ray structure of this complex previously described. It is important to clarify the solution behavior of these systems and, therefore, the binding of Hg(II) to HAH1 was probed over the pH range 7.5 to 9.4 using (199)Hg NMR, (199m)Hg PAC and UV-visible spectroscopies. The metal-dependent protein association over this pH range was examined using analytical gel-filtration. It can be concluded that at pH 7.5, Hg(II) is bound to a monomeric HAH1 as a two coordinate, linear complex (HgS2), like the Hg(II)-Atx1 X-ray structure (PDB ID: 1CC8). At pH 9.4, Hg(II) promotes HAH1 association, leading to formation of HgS3 and HgS4 complexes, which are in exchange on the μs-ns time scale. Thus, structures that may represent central intermediates in the process of metal ion transfer, as well as their exchange kinetics have been characterized.

Figure 1

¹⁹⁹Hg^II NMR spectra at various pH values, indicating a change in the coordination environment around the mercury(II).

Figure 2

Fourier transform (FT) of the experimental ^199mHg PAC data for HAH1 at pH 7.5 (solid line), 8.5 (dash line), and 9.4 (dash-dot line). The two major signals and their change with pH are indicated by the arrows. Samples contain 100 mM of the proper buffer, 200 μM HAH1, 100 μM of HgCl₂ and 55% w/w sucrose.

Figure 3

Electronic spectra of Hg^II complexes of HAH1 at different pH values (A) and comparison of spectra of Hg^II substituted rubredoxin at pH 8.0 (solid line) and Hg^II bound HAH1 at pH 9.4 (dotted line) (B). [HAH1] = 60 μM and [Hg^II] = 30 μM in 50 mM phosphate buffer at pH 6.5, 7.5, 8.5 and 50 mM CHES buffer at pH 9.4, respectively.

Figure 4

Gel filtration curves showing the elution profiles from a Superdex 75 HiLoad 16/600 column (pH range 7.5-9.4) for Hg^II-HAH1 (top) and apo-HAH1 (bottom). The top curve indicates that as the pH increases, Hg^II-HAH1 begins to dimerize. (Note: the shoulder appearing at ~85 mL at pH 9.4 in the Hg^II-HAH1 curve is likely apo-HAH1 monomer, since this is where the monomer elutes at this pH.) The same amount of apo-HAH1 and Hg^II-HAH1 was loaded for all injections. The increase in absorbance of apo-HAH1 at pH 9.4 may be due to the partial deprotonation of tyrosine.^[27] Samples for gel filtration experiments were prepared in 100 mM phosphate buffer, 200 mM NaCl, 1 mM TCEP for pH values 7.5 and 8.5, and 100 mM CHES, 200 mM NaCl, 1 mM TCEP for pH 9.4.

Figure 5

ESI-MS spectra of Hg^II complex of HAH1 at pH 6.5 in 1 mM carbonate buffer. [HAH1] = 1×10⁻⁴ M; Hg^II:HAH1 ratio 1:2; MeOH:H₂O = 50:50.

Figure 6

Generic graphical representation of Hg^II-HAH1 species formed at pH 7.5 (A) (linear HgS₂) and 9.4 (B) (equilibrium between T-shaped HgS₃ and 4-coordinate HgS₄ species). Figure 4B is not intended to specify the precise cysteinyl residues involved in metal binding. Protein backbone is shown as lines. Hg^II is shown as gray big sphere, Cys side chains are shown as ball and stick representations with sulfurs being shown as light gray small spheres. The figures were drawn in CS ChemOffice 2002 software package. Final figure conversion was made in Mercury.